Novel chemokine-like protein fragments

Abstract

This invention relates to TAFA proteins, fragments thereof, or variants thereof that have the ability to increase the length of neurites or branching points. Furthermore, this invention relates to pharmaceutical compositions containing polypeptides comprising the amino acid sequence of said TAFA protein, its fragments, or variants thereof; nucleic acid molecules encoding said polypeptides; vectors carrying said nucleic acid molecules; recombinant viral particles comprising said vector and capsid protein; cells comprising said vector; cells transformed with said vector; or therapeutic uses thereof. The compositions of this invention promote repair from damaged retina (e.g., cone cells of photoreceptors, etc.) or increase the length of neurites in neurons, and are therefore advantageous for the prevention or treatment of retinal neurological disorders and / or neuropathic pain.

Description

Technical Field

[0001] This disclosure relates to TAFA protein, TAFA protein fragments or variants thereof, which have the ability to increase neurite length and / or the number of branch points. Background Technology

[0002] The TAFA protein family is a group of proteins abundantly expressed in brain regions, consisting of five proteins: TAFA1 through TAFA5. These proteins contain structurally conserved cysteine ​​residues, and the C, CC, and CXC motifs between these cysteine ​​residues are associated with chemokines. TAFA1 through TAFA4 are highly similar in the number and spacing of cysteine ​​residues, while TAFA5 has even fewer cysteine ​​residues.

[0003] These proteins are highly conserved evolutionarily in vertebrates and have been shown to be important for the normal function of the central nervous system. In TAFA1 knockout mice, weight loss, reduced anxiety-like behaviors, and impaired fear memory have been reported (Lei X, Liu L, Terrillion CE et al., FASEB J., 2019, 33(12):14734-14747 and YongHJ, Ha N, Cho EB et al., Sci Rep., 2020, 10(1):3969). Conversely, when TAFA2 and TAFA3 are knocked out, increased anxiety-like behaviors have been reported (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, ShinHS, Sci Rep., 2017, 7(1):16503). In TAFA4-deficient mice, atypical pain and hyperalgesia have been reported (Delfini MC, Mantilleri A, Gaillard S et al., Cell Rep., 2013, 5(2):378-388). Behavioral changes, such as increased depressive-like behaviors and loss of spatial memory, have been reported in TAFA5 knockout mice (Huang S, Zheng C, Xie G et al., “FAM19A5 / TAFA5, a novel neurokine, plays acrucial role in depressive-like and spatial memory-related behaviors in mice”, Mol Psychiatry., 2021, 26(6):2363 2379).

[0004] Although these proteins play important roles in the central nervous system, our understanding of the function of each protein and its pathophysiological or therapeutic functions is very limited, and our understanding of their roles outside the central nervous system is also limited.

[0005] The above description of the background technology is only used to enhance the understanding of the background of the present invention and should not be construed as an admission that it is equivalent to the prior art known to those skilled in the art. Summary of the Invention

[0006] Technical issues

[0007] One object of this disclosure is to provide a polypeptide with the ability to increase neurite length and / or the number of branching points.

[0008] The polypeptide can reverse or restore the loss or dysfunction of cone cells, which are photoreceptor cells.

[0009] The polypeptide may contain the amino acid sequence of TAFA (TAFA chemokine-like family member) protein, fragments thereof, or variants thereof.

[0010] The polypeptide may contain the following amino acid sequence of general formula 7 from the N-terminus to the C-terminus:

[0011] General Formula 7:

[0012]

[0013] In general formula 1,

[0014] X1 does not exist, or is V, I, or L;

[0015] X2 is K, E, R, or Q;

[0016] X3 can be G, T, Q, P, or A;

[0017] X4 is either V or I;

[0018] X5 can be A, L, V, or I;

[0019] X6 is either R or L;

[0020] X7 is K, R, or Q;

[0021] X8 is either R or K;

[0022] X9 is either R or L;

[0023] X10 is either V or G;

[0024] X11 is either K or N;

[0025] X12 is either F or L;

[0026] X13 is either P or S;

[0027] X14 is either Q or K;

[0028] X15 is R, H, or Q;

[0029] X16 can be A, N, S, or T;

[0030] X17 is A, Q, R, K, or T;

[0031] X18 is either D or E;

[0032] X19 is either S or A;

[0033] X20 can be I, E, L, A, or V;

[0034] X21 is Q, G, or E;

[0035] X22 is either K or R;

[0036] X23 is H, Q, or E;

[0037] X24 can be E, Q, N, D, S, or H;

[0038] X25 is available in L, V, or M sizes;

[0039] X26 can be E, D, P, L, or A;

[0040] X27 is either E or D;

[0041] X28 can be V, T, A, or I;

[0042] X29 can be L, N, R, Y, S, or Q;

[0043] X30 is available in S, K, or T;

[0044] X31 is either S or M;

[0045] X32 can be S, A, or Y;

[0046] X33 can be S, T, or R;

[0047] X34 is either N or H;

[0048] X35 is either V or I;

[0049] X36 is either R or K;

[0050] X37 does not exist, or is V, A, G, M, or N;

[0051] X38 does not exist, or is T, I, N, F, or S; and

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

[0053] The polypeptide may comprise one or more amino acid sequences selected from the amino acid sequences shown in SEQ ID NO: 87 to SEQ ID NO: 141.

[0054] The polypeptide can consist of 8 to 61 amino acid residues.

[0055] The polypeptide may contain an amino acid sequence that has at least 50% sequence identity with the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 15 or SEQ ID NO: 87.

[0056] The amino acid sequence of the polypeptide can include the following amino acid sequence of general formula 1 from the N-terminus to the C-terminus:

[0057] General Formula 1:

[0058]

[0059] In general formula 1,

[0060] X1 can be E, D, P, L, or A;

[0061] X2 is either D or E; and

[0062] X3 can be T, V, I, or A.

[0063] The amino acid sequence of the polypeptide comprising the amino acid sequence of general formula 1 may comprise the amino acid sequence of SEQ ID NO: 142.

[0064] The amino acid sequence of the polypeptide comprising the amino acid sequence of general formula 1 may comprise an amino acid sequence selected from the amino acid sequences of SEQ ID NO: 1 to SEQ ID NO: 14.

[0065] The amino acid sequence of the polypeptide comprising the amino acid sequence of general formula 1 may consist of 8 to 43 amino acid residues.

[0066] The amino acid sequence of the polypeptide can include the following amino acid sequence of general formula 2 from the N-terminus to the C-terminus:

[0067] General Formula 2:

[0068]

[0069] In general formula 2,

[0070] X1 can be E, D, P, L, or A;

[0071] X2 is either D or E;

[0072] X3 can be T, V, I, or A;

[0073] X4 can be I, E, A, L, or V;

[0074] X5 is Q, E, or G;

[0075] X6 is either K or R;

[0076] X7 is E, H, or Q;

[0077] X8 can be E, Q, N, D, S, or H; and

[0078] X9 can be L, M, or V.

[0079] The amino acid sequence of the polypeptide comprising the amino acid sequence of general formula 2 may comprise the amino acid sequence of SEQ ID NO: 143. The amino acid sequence of the polypeptide comprising the amino acid sequence of general formula 2 may comprise an amino acid sequence selected from the amino acid sequences of SEQ ID NO: 28 to SEQ ID NO: 51.

[0080] The amino acid sequence of the polypeptide can include the following amino acid sequence of general formula 3 from the N-terminus to the C-terminus:

[0081] General Formula 3:

[0082]

[0083] In general formula 3,

[0084] X1 can be E, D, P, L, or A;

[0085] X2 is either D or E;

[0086] X3 can be T, V, I, or A;

[0087] X4 can be Y, S, or L;

[0088] X5 is either S or T;

[0089] X6 is either S or T; and

[0090] X7 is either V or I.

[0091] The amino acid sequence of the polypeptide comprising the amino acid sequence of formula 3 may comprise the amino acid sequence of SEQ ID NO: 144.

[0092] The amino acid sequence of the polypeptide comprising the amino acid sequence of general formula 3 may comprise an amino acid sequence selected from the amino acid sequences of SEQ ID NO: 52 to SEQ ID NO: 58.

[0093] The amino acid sequence of the polypeptide may include one or more amino acid sequences selected from SEQ ID NO: 152 to SEQ ID NO: 171.

[0094] The amino acid sequence of the polypeptide can include the following amino acid sequence of general formula 4 from the N-terminus to the C-terminus:

[0095] Formula 4:

[0096]

[0097] In general formula 4,

[0098] X1 is either Q or K;

[0099] X2 is R, H, or Q;

[0100] X3 can be A, N, S, or T;

[0101] X4 can be R, A, Q, K, or T;

[0102] X5 is either D or E;

[0103] X6 is either A or does not exist; and

[0104] X7 is S, A, or does not exist.

[0105] The amino acid sequence of the polypeptide comprising the amino acid sequence of formula 4 may comprise the amino acid sequence of SEQ ID NO: 145.

[0106] The amino acid sequence of the polypeptide comprising the amino acid sequence of general formula 4 may comprise an amino acid sequence selected from the amino acid sequences of SEQ ID NO: 15 to SEQ ID NO: 27.

[0107] The amino acid sequence of the polypeptide comprising the amino acid sequence of general formula 4 may consist of 15 to 46 amino acid residues.

[0108] The amino acid sequence of the polypeptide can include the following amino acid sequence of general formula 5 from the N-terminus to the C-terminus:

[0109] Formula 5:

[0110]

[0111] In general formula 5,

[0112] X1 is either Q or K;

[0113] X2 is R, H, or Q;

[0114] X3 can be A, N, S, or T;

[0115] X4 can be R, A, Q, K, or T;

[0116] X5 is either D or E;

[0117] X6 is either A or does not exist;

[0118] X7 is S, A, or does not exist;

[0119] X8 is either R or K;

[0120] X9 is either R or L;

[0121] X10 is either V or G;

[0122] X11 is either K or N;

[0123] X12 is either F or L; and

[0124] X13 is either P or S.

[0125] The amino acid sequence of the polypeptide comprising the amino acid sequence of formula 5 may comprise the amino acid sequence of SEQ ID NO: 146.

[0126] The amino acid sequence of the polypeptide comprising the amino acid sequence of formula 5 may comprise an amino acid sequence selected from the amino acid sequences of SEQ ID NO: 59 to SEQ ID NO: 74.

[0127] The amino acid sequence of the polypeptide can include the following amino acid sequence of general formula 6 from the N-terminus to the C-terminus:

[0128] Formula 6:

[0129]

[0130] In general formula 6,

[0131] X1 is either Q or K;

[0132] X2 is R, H, or Q;

[0133] X3 can be A, N, S, or T;

[0134] X4 can be R, A, Q, K, or T;

[0135] X5 is either D or E;

[0136] X6 is either A or does not exist;

[0137] X7 is S, A, or does not exist;

[0138] X8 can be I, A, V, or L;

[0139] X9 is either Q or E;

[0140] X10 is either H or Q;

[0141] X11 is N, D, S, or H; and

[0142] X12 is either L or M.

[0143] The amino acid sequence of the polypeptide comprising the amino acid sequence of formula 6 may comprise the amino acid sequence of SEQ ID NO: 147.

[0144] The amino acid sequence of the polypeptide comprising the amino acid sequence of general formula 6 may consist of 75 to 85 amino acid residues.

[0145] The amino acid sequence of the polypeptide comprising the amino acid sequence of formula 6 may comprise one or more amino acid sequences selected from SEQ ID NO: 172 to SEQ ID NO: 184.

[0146] Another object of this disclosure is to provide a nucleic acid molecule encoding the said polypeptide.

[0147] Another object of this disclosure is to provide a vector comprising the said nucleic acid molecule.

[0148] Another object of this disclosure is to provide recombinant viral particles comprising the said vector and capsid protein.

[0149] The virus may be AAV.

[0150] Another object of this disclosure is to provide cells comprising the said vector.

[0151] Another object of this disclosure is to provide cells transformed using the said vector.

[0152] Another object of this disclosure is to provide a composition comprising the polypeptide, a nucleic acid molecule encoding the polypeptide, a vector comprising the nucleic acid molecule, a recombinant viral particle comprising the vector and a capsid protein, a cell comprising the vector, a cell transformed with the vector, or any combination thereof.

[0153] The composition may be a pharmaceutical composition.

[0154] The pharmaceutical composition may be a composition for the prevention or treatment of retinal neurodegenerative diseases.

[0155] The retinal neurodegenerative diseases may include retinopathy, choroidal neovascularization, macular degeneration, macular degeneration, retinal degeneration, macular edema, retinal swelling, macular swelling, retinal swelling, color vision deficiency, retinal cell degeneration, retinal vascular occlusion, retinal detachment, hereditary retinal diseases, or any combination thereof.

[0156] The pharmaceutical composition may be a composition for the prevention or treatment of neuropathic pain.

[0157] The neuropathic pain can be allodynia, hyperalgesia, hyperesthesia, or dysesthesia.

[0158] The neuropathic pain can be central or peripheral.

[0159] The neuropathic pain can be neuralgia, deafferentation pain syndrome, complex regional pain syndrome, or (central or peripheral) neuropathy.

[0160] Another object of this disclosure is to provide a method for producing a composition comprising the steps of producing the composition, said composition comprising the polypeptide, a nucleic acid molecule encoding the polypeptide, a vector comprising the nucleic acid molecule, a recombinant viral particle comprising the vector and a capsid protein, a cell comprising the vector, a cell transformed with the vector, or any combination thereof.

[0161] Another object of this disclosure is to provide the therapeutic use of the said polypeptide, the nucleic acid molecule encoding the said polypeptide, the vector comprising the nucleic acid molecule, the recombinant viral particle comprising the vector and the capsid protein, the cell comprising the vector, the cell transformed with the vector, or any combination thereof, in the preparation of a medicament.

[0162] Another object of this disclosure is to provide a method for preventing or treating a disease or condition in a subject in need, the method comprising administering a composition to the subject, the composition comprising the polypeptide, a nucleic acid molecule encoding the polypeptide, a carrier comprising the nucleic acid molecule, a recombinant viral particle comprising the carrier and a capsid protein, a cell comprising the carrier, a cell transformed with the carrier, or any combination thereof.

[0163] Other objects and advantages of the present invention will become more apparent from the following detailed description of the invention, the claims and the accompanying drawings.

[0164] Technical solution

[0165] I. Definition

[0166] Throughout this disclosure, the term "at least" preceding a single number or a series of numbers is understood to include the number following the term "at least" as well as all subsequent numbers or integers that may logically be included depending on the context. For example, the number of nucleotides in a nucleic acid molecule must be an integer. Therefore, "at least 18 nucleotides of a nucleic acid molecule of 21 nucleotides" means that 18, 19, 20, or 21 nucleotides have the specified properties. When "at least" appears before a series of numbers or a range, it should be understood that "at least" may modify each number in that series or range. "At least" is also not limited to integers; for example, "at least 5%" includes 5.0%, 5.1%, and 5.18%, regardless of the number of significant figures.

[0167] Where the term “comprising” is used to describe aspects in this specification, it should be understood that other similar aspects described in terms of “consisting of” and / or “substantially consisting of” are also provided.

[0168] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. For example, the Concise Dictionary of Biomedicine and Molecular Biology, Juo, Pei Show, 2nd ed., 2002, CRC Press; Dictionary of Cell and Molecular Biology, 3rd ed., 1999, Academic Press; and Oxford Dictionary of Biochemistry and Molecular Biology, Revised, 2000, Oxford University Press provide a general dictionary of many terms used in this disclosure for those skilled in the art.

[0169] Units, prefixes, and symbols are represented in the form accepted by Systeme International de Unites (SI). Numerical ranges include the numbers defining the range. Unless otherwise stated, amino acid sequences are written from left to right, from amino to carboxyl. The headings provided herein are not intended to limit the various aspects of this disclosure, which can be obtained by referring to the entire specification. Therefore, the terms defined below are defined more fully by referring to the entire specification.

[0170] As used herein, the term “about” means approximately, roughly, around, or within a range of. When the term “about” is used in conjunction with a numerical range, it modifies the range by extending the boundaries above and below the stated value. Typically, the term “about” can modify a numerical value to be above and below the stated value, for example, a deviation of 10%, a deviation above 10%, or a deviation below 10%.

[0171] As used herein, the terms “sequence-similar family 19” or “FAM19” or “TAFA” refer to proteins belonging to the TAFA family (also known as the FAM19 family), which are primarily expressed in the brain and spinal cord. FAM19A1 is also known as TAFA1, FAM19A2 is also known as TAFA2, FAM19A3 is also known as TAFA3, FAM19A4 is also known as TAFA4, and FAM19A5 is also known as TAFA5.

[0172] The human TAFA1 gene encodes a 133-amino acid sequence. Although there is disagreement regarding the length of the signal peptide and the mature protein, the human TAFA1 protein is expected to consist of a 35-amino acid signal sequence and a 98-amino acid mature protein. TAFA1 is highly expressed in the frontal, temporal, occipital, and parietal cortices, and at lower levels in the basal ganglia, lateral canal, and cerebellum. Cellular experiments have shown that TAFA1 influences the determination of neural stem cell differentiation fate, inhibiting the differentiation of neural stem cells into astrocytes and promoting their differentiation into neurons. Experiments using TAFA1 knockout (KO) mice have shown that TAFA1 can regulate voluntary activity, anxiety-like behavior, learning and memory, and somatosensory functions.

[0173] The human TAFA2 gene encodes a 131-amino acid sequence. Although there is disagreement regarding the length of the signal peptide and the mature protein, the human TAFA2 protein is expected to consist of a 30-amino acid signal sequence and a 101-amino acid mature protein. TAFA2 is most abundantly expressed in the occipital cortex, frontal cortex, and medulla oblongata of the central nervous system. It is known that injecting recombinant TAFA2 protein into the third ventricle of mice increases food intake and feeding frequency, energy expenditure, and respiratory exchange rate. This suggests that TAFA2 may play a role in regulating food intake and energy metabolism. It is also known that inhibiting TAFA2 in zebrafish and mice increases anxiety-like behaviors.

[0174] The human TAFA3 gene encodes a 133-amino acid sequence. Although there is disagreement regarding the length of the signal peptide and the mature protein, the human TAFA3 protein is expected to consist of a 30-amino acid signal sequence and a 103-amino acid mature protein. It is known that TAFA3 expression is increased in microglia in mouse models of transient focal cerebral ischemia, and microglia treated with TAFA3 polarize into anti-inflammatory microglia. Furthermore, when TAFA3 is knocked out in mouse models, three major behavioral deficits are observed in autism spectrum disorder: reduced responsiveness to social novelty, impaired social communication, and increased repetitive behaviors. This suggests that TAFA3 plays a role in the normal functioning of social relationship formation.

[0175] The human TAFA4 gene encodes a 140-amino acid sequence. Although there is disagreement regarding the length of the signal peptide and the mature protein, the human TAFA4 protein is expected to consist of a 45-amino acid signal sequence and a 95-amino acid mature protein. TAFA4 is primarily expressed in sensory neurons of the peripheral nervous system. The TAFA4 protein is specifically expressed in low-threshold mechanoreceptors and appears to alleviate pain by modulating the activity of interneurons, particularly GABAergic neurons.

[0176] The human TAFA5 gene encodes a 132-amino acid sequence. Although there is disagreement regarding the lengths of the signal peptide and the mature protein, the human TAFA5 protein is expected to consist of a 43-amino acid signal peptide and an 89-amino acid mature protein. TAFA5 is highly expressed in the basal ganglia region and the cerebellum. Inflammatory stimuli such as TNF-α are known to increase TAFA5 expression in the mouse hypothalamus, and when TAFA5 is knocked out, TNF-α-induced suppression of food intake, weight loss, and increased inflammatory cytokines are partially reversed.

[0177] For the mature proteins TAFA1 to TAFA4, they exhibit high sequence identity (e.g., human TAFA4 shares 73.7% sequence identity with TAFA1, 85.3% with TAFA2, and 81.1% with TAFA3). On the other hand, TAFA5 exhibits low sequence identity with TAFA1 to TAFA4 (e.g., human TAFA5 shares 48.9% sequence identity with TAFA1, 51.1% with TAFA2, 47.7% with TAFA3, and 50.0% with TAFA4), and is therefore expected to have a completely different function.

[0178] Human TAFA4 (TAFA chemokine-like family member 4) protein (SEQ ID NO 299) is a member of the TAFA protein family (TAFA1 to TAFA5), which is richly expressed in brain regions. Composed of 140 amino acids, TAFA4 has 95 or 93 amino acids, excluding the first 45 or 47 amino acids, which is highly conserved evolutionarily in vertebrates. Furthermore, within the TAFA proteome, TAFA1 to TAFA4 exhibit highly conserved interspecific sequence identity within 93 amino acid sequences in vertebrates. i) The mature human TAFA4 amino acid sequence shows 90% or higher sequence identity compared to TAFA4 in mammals and amphibians, 95% or higher sequence identity compared to TAFA4 in birds, and 85% or higher sequence identity compared to TAFA4 in reptiles and fish. ii) The human TAFA1 amino acid sequence shows 90% or higher sequence identity compared to TAFA1 in mammals and amphibians, 95% or higher sequence identity compared to TAFA1 in birds, and 85% or higher sequence identity compared to TAFA1 in reptiles and fish. iii) The human TAFA2 amino acid sequence shows 90% or higher sequence identity compared to TAFA2 in mammals and amphibians, 95% or higher sequence identity compared to TAFA2 in birds, and 80% or higher sequence identity compared to TAFA2 in reptiles and fish. Furthermore, iv) The human TAFA3 amino acid sequence shows 75% or higher sequence identity compared to TAFA3 in mammals and amphibians, 80% or higher sequence identity compared to TAFA3 in birds, and 75% or higher sequence identity compared to TAFA3 in reptiles and fish. It can be seen that they exhibit very high sequence identity, regardless of species.

[0179] As used herein, the term "TAFA protein fragment" refers to fragments of TAFA1 through TAFA4 proteins that have the ability to increase neurite length and / or the number of branching points. These fragments may consist of 8 (e.g., TAFA fragment 3.7), 9, 10, 11, 12, 13, 14, 15, 16, 17 (e.g., TAFA fragment 2.5), 18, 19, 20, 21, 22 (e.g., TAFA fragment 7), 23, 24, 25 (e.g., TAFA fragment 2.5+3.7), 26, 27, 28, 29 (e.g., TAFA fragment 3), 30, 31 (e.g., TAFA fragment 2), or 32 (e.g., TAFA fragment 5). It consists of 33, 34, 35, 36, 37, 38, 39 (e.g., TAFA fragment 2.5+7 or TAFA fragment 2+3.7), 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54 (e.g., TAFA fragment 5+7), 55, 56, 57, 58, 59, 60 (e.g., TAFA fragment 2+3) or 61 (e.g., TAFA fragment 5+3) amino acid residues, but is not limited thereto.

[0180] As used herein, the term "variant of a TAFA protein fragment" refers to a polypeptide in which some amino acids of the polypeptide constituting the TAFA1 to TAFA4 protein fragments are substituted with other amino acids. The variant is preferably a functional variant. The term "functional" refers to a variant derived from the fragments of the TAFA1 to TAFA4 proteins that, like the "TAFA protein fragments," has the ability to increase neurite length and / or the number of branching points. The variant is preferably an interspecies variant (e.g., a polypeptide containing any one of the amino acid sequences in SEQ ID NO: 142 to SEQ ID NO: 147). Those skilled in the art can readily prepare polypeptides in which one or more amino acids are substituted with other amino acids by excluding non-conserved sequence differences from the amino acid sequences of the TAFA1 to TAFA4 fragments of each known species. For example, a polypeptide can be generated by replacing the fourth residue D with E in the human TAFA4 fragment 3.7 sequence EGEDCKVL (SEQ ID NO: 1) by referring to the rabbit TAFA2 fragment 3.7 sequence EGEECKVL (SEQ ID NO: 6), or by replacing the first residue E with P and the fourth residue D with E by referring to the porcine TAFA3 fragment 3.7 sequence PGEECKVL (SEQ ID NO: 9).

[0181] The number of amino acid substitutions can range from 1 to 22 amino acids. For example, when a TAFA protein fragment contains both fragment 5 and fragment 7, 1 to 22 amino acid substitutions can be performed; when a TAFA protein fragment contains both fragment 2 and fragment 3, 1 to 19 amino acid substitutions can be performed; when a TAFA protein fragment contains only fragment 2, 1 to 12 amino acid substitutions can be performed; when a TAFA protein fragment contains only fragment 5, 1 to 13 amino acid substitutions can be performed; when a TAFA protein fragment contains only fragment 3, 1 to 7 amino acid substitutions can be performed; when a TAFA protein fragment contains only fragment 7, 1 to 9 amino acid substitutions can be performed; when a TAFA protein fragment contains only fragment 2.5, 1 to 7 amino acid substitutions can be performed; and when a TAFA protein fragment contains only fragment 3.7, 1 to 3 amino acid substitutions can be performed.

[0182] Preferably, the number of amino acid substitutions is 1 to 20, 1 to 19, 1 to 18, 1 to 17, 1 to 16, 1 to 15, 1 to 14, 1 to 13, 1 to 12, 1 to 11, 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4 or 1 to 3.

[0183] As used herein, the term "adeno-associated virus" or "AAV" refers to a single-stranded DNA virus that is a helper-dependent human parvovirus. The genome is approximately 4.6 kbp in size. The N-terminal portion of the genome encodes the rep gene, which is involved in viral replication and viral gene expression, while the C-terminal portion encodes the cap gene, which encodes the viral capsid protein. Both ends contain intercalary repeats (ITRs) of approximately 145 bases each. The T-shaped, 145 bp ITRs act as the origin of replication during viral genome replication and serve as the primary packaging signal. Since the ITR is the only cis-acting nucleotide sequence required for the preparation of recombinant AAV constructs and exhibits enhancer activity in the presence of the Rep protein but very weak activity in its absence, it is necessary to take this into account when cloning transgenes into recombinant AAV constructs and to construct expression constructs by appropriately arranging enhancers, promoters, pA, etc. (RJ Samulski and N Muzyczka, Annu. Rev. Virol., 2014, 1:427-451). Four proteins, classified by molecular weight as rep78, rep68, rep52, and rep40, are translated from the rep gene and perform important functions in AAV DNA replication. These four proteins are translated from the cap gene; VP1, VP2, and VP3 are structural proteins that make up AAV particles, and the assembly activation protein (AAP) promotes AAV particle formation through these structural proteins. For efficient adeno-associated virus replication, certain 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).

[0184] AAVs include, but are not limited to, AAV 1, AAV 2, AAV 3 (including 3A and 3B), AAV 4, AAV 5, AAV 6, AAV 7, AAV 8, AAV 9, AAV 10, AAV 11, AAV 12, AAV 13, AAVrh.74, snake AAVs, bird AAVs, cattle AAVs, dog AAVs, horse AAVs, sheep AAVs, goat AAVs, shrimp AAVs, those AAV serotypes and clades disclosed by Gao et al. (J. Virol., 78:6381 (2004)) and Moris et al. (Virol., 33:375 (2004)), and any other AAVs now known or discovered later. See, for example, FIELDS et al., VIROLOGY, Vol. 2, Chapter 69, 4th ed., Lippincott-Raven Publishers. In some respects, "AAV" includes derivatives of known AAV. In some respects, "AAV" includes modified or artificial AAV. In some respects, "AAV" includes AAV having a modified capsid.

[0185] As used herein, the terms “application,” “administration,” and variations thereof refer to the introduction of a composition (e.g., a polypeptide of the amino acid sequence of a TAFA protein, fragment thereof, or variant thereof, as described herein) into a subject via a pharmaceutically acceptable route. The composition is introduced into the subject via any suitable route, including intratumoral, oral, pulmonary, intranasal, non-enteric (e.g., intravenous, intraarterial, intramuscular, intraperitoneal, or subcutaneous), rectal, intralymphatic, intrathecal, periocular, intraocular, or topical (e.g., eye drops, nasal administration, etc.). Application includes self-application and administration by another person. The composition or formulation exerts its intended function through a suitable route of administration. For example, if the suitable route is intravenous, the composition is administered by introducing the composition or formulation into the subject's vein. Pharmaceutical compositions may be formulated for topical or local application, such as as eye drops, gels, creams, or lotions for application to the eyes, skin, or mucous membranes, and for intrathecal or intracisional administration. Local application may be considered for ocular, transdermal, or mucosal (e.g., nasal, oral, rectal, etc.) or for inhalation therapy. The composition can be applied alone or in combination with other pharmaceutically acceptable excipients.

[0186] As used herein, the term "intraocular" means within or beneath the ocular tissues. As used herein, the term "intraocular administration" means any administration capable of delivering the composition to a sub-Tenon site, subconjunctival site, suprachoroidal site, subretinal site, vitreous site, or similar site in the eye. In some aspects, intraocular administration includes suprachoroidal, subretinal, and vitreous administration.

[0187] As used herein, the term "conservative" means that each nucleotide or amino acid residue in a polynucleotide or polypeptide sequence remains unchanged at the same position in each of two or more sequences being compared. Relatively conserved nucleotides or amino acids are those that are conserved in the relevant sequence, rather than other nucleotides or amino acids that appear at other positions in the sequence.

[0188] As used herein, the term "amino acid" includes not only the 20 standard amino acids typically incorporated into peptides (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), and cysteine ​​(C)), but also D-isomers and modified amino acids. Furthermore, peptides may contain non-standard amino acids that have undergone post-translational modifications. Post-translational modifications may include, but are not limited to, phosphorylation, glycosylation, acylation (e.g., acetylation, myristylation, and palmitoylation), alkylation, carboxylation, hydroxylation, glycosylation, biotinylation, ubiquitination, changes in chemical properties (e.g., β-elimination deiminolation and deamidation), and structural changes (e.g., disulfide bond formation). Peptides can be wild-type peptides identified and isolated from natural sources. Alternatively, peptides can be artificial variants comprising an amino acid sequence in which one or more amino acids are substituted, deleted, and / or inserted. Amino acid modifications in artificial variants and wild-type peptides can include conserved amino acid substitutions that do not significantly affect protein folding and / or activity. For example, such conserved substitutions may occur between basic amino acids (arginine (R), lysine (K), and histidine (H)), acidic amino acids (glutamate (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)). Generally, amino acid substitutions that do not alter specific activity are known in the art. The most common substitutions can include Ala / Ser, Val / Ile, Asp / Glu, Thr / Ser, Ala / Gly, Ala / Thr, Ser / Asn, Ala / Val, Ser / Gly, Tyr / Phe, Ala / Pro, Lys / Arg, Asp / Asn, Leu / Ile, Leu / Val, Ala / Glu, and Asp / Gly.

[0189] In some respects, when two or more sequences are 100% identical to each other, they are called "completely conserved" or "identical." In some respects, when two or more sequences are at least 70%, at least 80%, at least 90%, or at least 95% identical to each other, they are called "highly conserved." In some respects, when two or more sequences are approximately 70%, approximately 80%, approximately 90%, approximately 95%, approximately 98%, or approximately 99% identical to each other, they are called "highly conserved." In some respects, when two or more sequences are at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% identical to each other, they are called "conservative." In some respects, two or more sequences are said to be "conserved" when they share approximately 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, or 99% identity with each other. Sequence conservation can apply to the entire length of a polynucleotide or polypeptide, or to its parts, regions, or features.

[0190] The terms “complementary” and “complementarity” refer to the relationship between two or more oligomers, each containing a nucleotide sequence, or between an oligomer and a target gene, as determined by the Watson-Crick base pairing rules. For example, the nucleotide sequence “TGA (5′→3′)” is complementary to the nucleotide sequence “ACT (3′→5′)”. Complementarity can be “partial” when all bases in a given nucleotide sequence match fewer bases than those in another nucleotide sequence according to the base pairing rules. For example, in some respects, 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%. Thus, in some respects, the term “complementary” refers to at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity or complementarity with the target nucleic acid sequence. Alternatively, continuing this example, a given nucleotide sequence can have “complete” or “perfect” (100%) complementarity with another nucleotide sequence. In some respects, the degree of complementarity between nucleotide sequences has a substantial impact on the efficiency and strength of hybridization between sequences.

[0191] The term "downstream" refers to the nucleotide sequence located 3' to the left of the reference nucleotide sequence. In some respects, downstream nucleotide sequences are associated with sequences located after the transcription start site. For example, the translation start codon of a gene is located downstream of the transcription start site.

[0192] As used herein, the term "enhancer" refers to a portion of DNA that contains sequences capable of providing enhanced transcription and, in some cases, can function independently of its orientation relative to another regulatory sequence. Enhancers can work in conjunction with promoters and / or other enhancer elements or function additionally.

[0193] The terms “excipient” and “carrier” are used interchangeably to refer to an inert substance added to a pharmaceutical composition to facilitate the administration of a compound (e.g., a polynucleotide containing a transgene and an untranslated nucleic acid sequence as described herein).

[0194] The term "exon" refers to a specific portion of a nucleic acid that encodes a protein, or the nucleic acid sequence that appears in the mature form of an RNA molecule after removal through splicing of portions of pre-processed or precursor RNA. Mature RNA molecules can be functional forms of messenger RNA (mRNA) or non-coding RNA such as rRNA or tRNA.

[0195] As used herein, the term "expression" refers to the process by which polynucleotides produce gene products (such as RNA or polypeptides). This includes, but is not limited to, the transcription of polynucleotides into messenger RNA (mRNA) and the translation of mRNA into polypeptides. Expression produces "gene products."

[0196] As used herein, gene products can be nucleic acids, such as RNA produced through gene transcription. Gene products as used herein can be nucleic acids or polypeptides translated from transcripts. Gene products described herein also include nucleic acids that have undergone post-transcriptional modifications (e.g., polyadenylation or splicing) or polypeptides that have undergone post-translational modifications (e.g., phosphorylation, methylation, glycosylation, lipid addition, association with other protein subunits, or proteolytic cleavage of proteins).

[0197] As used herein, the term "identity" refers to the overall monomer conservation between polymer molecules (e.g., between polynucleotide molecules). The term "identical" when used without additional modifiers, such as "polynucleotide A is identical to polynucleotide B," means that the polynucleotide sequences are 100% identical (i.e., 100% sequence identity). For example, describing two sequences as "70% identical" is the same as describing them as having "70% sequence identity."

[0198] For example, the percentage of identity between two polypeptide or polynucleotide sequences can be calculated by aligning the two sequences to obtain the optimal comparison. For optimal alignment, vacancies can be introduced in one or both of the first and second polypeptide or polynucleotide sequences, and mismatched sequences can be ignored for comparison purposes. In some respects, the length of the alignment sequence used for comparison 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 of the polypeptide at the corresponding positions, or the bases of the polynucleotide at the corresponding positions, are then compared.

[0199] When the same amino acid or nucleotide occupies a specific position in a first sequence and the corresponding position in a second sequence, the molecule is identical at that position. The percentage identity between two sequences is a function of the number of positions of the same residues shared by the two sequences, taking into account the number of vacancies that must be introduced to achieve optimal alignment of the two sequences and the length of each such vacancy. Sequence comparisons and percentage identity determination between two sequences can be performed using mathematical algorithms.

[0200] Suitable software programs for aligning different sequences (e.g., polynucleotide sequences) are available from various sources. One suitable program for determining sequence identity (percentage) is bl2seq, which is part of the BLAST program suite available from the National Center for Biotechnology Information (NCBI) website (blast.ncbi.nlm.nih.gov). bl2seq uses either the BLASTN or BLASTP algorithm for paired sequence comparison. BLASTN is used to compare nucleic acid sequences, while BLASTP is used to compare amino acid sequences. Other suitable programs include, for example, Needle, Stretcher, Water, or Matcher, which are part of the EMBOSS bioinformatics program suite and are also available from the European Institute for Bioinformatics (EBI) at www.ebi.ac.uk / Tools / psa.

[0201] Sequence alignment can be performed using methods known in the art, such as MAFFT, Clustal (e.g., ClustalW, ClustalX, or Clustal Omega), and MUSCLE.

[0202] Different regions within a single polynucleotide or polypeptide target sequence aligned to a reference polynucleotide or polypeptide sequence can each have their own percentage sequence identity. Note that percentage sequence identity values ​​are rounded to the nearest decimal place. 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 ​​are always integers.

[0203] In some respects, the percentage identity (%ID) between the first amino acid sequence (or nucleic acid sequence) and the second amino acid sequence (or nucleic acid sequence) is calculated as %ID = 100 × (Y / Z), where Y is the number of amino acid residues (or nucleotides) counted as identical matches in the alignment of the first and second sequences (e.g., determined by visual inspection or using a specific sequence alignment procedure), and Z is the total number of residues in the second sequence. If the first sequence is longer than the second sequence, the percentage identity of the first sequence to the second sequence will be higher than the percentage identity of the second sequence to the first sequence.

[0204] Those skilled in the art will understand that the generation of sequence alignments used to calculate sequence identity (percentage) is not limited to comparisons of binary sequences considering only primary sequence data. It will also be understood that sequence alignments can be generated by integrating sequence data with data from heterogeneous sources, such as structural data (e.g., crystallographic protein structures), functional data (e.g., mutation locations), or phylogenetic data. A suitable program for generating multiple sequence alignments by integrating heterogeneous data is T-Coffee, available from www.tcoffee.org and, for example, EBI. It will also be understood that the final alignments used to calculate sequence identity (percentage) can be arranged automatically or manually.

[0205] As used herein, the term "intron" refers to an intercalated portion of DNA within a gene that does not encode the protein produced by that gene and is spliced ​​out of the mRNA transcribed from that gene before being exported from the nucleus. The term "intronic sequence" refers to the nucleic acid sequence of an intron. Such sequences are also referred to herein as "untranslated nucleic acid sequences." Thus, an intron is a region of DNA that is transcribed along with the coding exon but is removed during the formation of mature mRNA.

[0206] As used herein, the term "intronic fragment" refers to a fragment derived from a full-length intron sequence (e.g., a full-length EF-1α intron A sequence). The fragment excludes full-length introns. In some aspects, an "intronic fragment" comprises the minimum number or configuration of nucleotides required to achieve an expression level exceeding that achieved by a corresponding construct in which all nucleotides of EF-1α intron A are absent. Therefore, the intronic fragments of this disclosure are also referred to herein as "non-translated nucleic acid sequences" without particular limitation, provided they contain fragments of EF-1α introns and can increase transgene expression. As demonstrated herein, in some aspects, intronic fragments (i.e., non-translated nucleic acid sequences) can increase transgene transcription, thereby increasing transgene expression. Therefore, in some aspects, the intronic fragments described herein can be non-translated regulatory elements.

[0207] As used herein, the terms “isolated,” “purified,” “extracted,” and variations thereof are used interchangeably and refer to the state of preparation of the compositions of this disclosure, such as polypeptides comprising the amino acid sequence of a TAFA protein, a fragment thereof, or a variant thereof, or polynucleotides comprising the nucleic acid sequence encoding such a protein, having undergone one or more purification steps. In some aspects, isolation or purification as used herein refers to the process of removing (e.g., fractionating) the compositions of this disclosure, such as the polypeptides or polynucleotides described herein, from a sample containing impurities.

[0208] In some respects, the isolated composition has no detectable undesirable activity, or alternatively has a level or amount of undesirable activity at or below an acceptable level or amount. In other respects, the isolated composition has an amount and / or concentration and / or activity higher than that of the desired composition of this disclosure. In other respects, the isolated composition is enriched relative to the starting material from which the composition was obtained. Such enrichment relative to the starting material may 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%.

[0209] In some respects, the isolated preparation is substantially free of residual biological products. In some respects, the isolated preparation does not contain 100%, at least about 99%, at least about 98%, at least about 97%, at least about 96%, at least about 95%, at least about 95%, at least about 94%, at least about 93%, at least about 92%, at least about 91%, or at least about 90% of biological contaminants. Residual biological products may include non-biological substances, including chemical substances or undesirable nucleic acids, proteins, lipids, or metabolites.

[0210] As used herein, the term "linked" refers to a first amino acid or polynucleotide sequence covalently or non-covalently linked to a second amino acid or polynucleotide sequence. The first amino acid or polynucleotide sequence may be directly bonded to or adjacent to the second amino acid or polynucleotide sequence, or alternatively, an intercalation sequence may covalently link the first sequence to the second sequence. For example, since the TAFA protein is a polypeptide comprising fragments 1, 2, and 3 linked together, the fragments of the TAFA protein may be in the form of fragment 2 linked to fragment 3, or fragment 5 linked to fragment 7. The term "linked" not only means fusing a first polynucleotide sequence to the 5' or 3' end of a second polynucleotide sequence, but also includes inserting the entire first polynucleotide sequence or the entire second polynucleotide sequence between any two nucleotides within the second or first polynucleotide sequence, respectively. The first polynucleotide sequence may be linked to the second polynucleotide sequence via a phosphodiester bond or via a linker. The linker may be, for example, a polynucleotide.

[0211] As used herein, the term "retinal neurodegenerative disease" refers to any disease, condition, or condition that affects or is associated with the nerve portion or region of the retina or macula of the eye. Retinal neurodegenerative diseases can be caused by damage to all or part of the nerves in the retina or macula. Retinal neurodegenerative diseases can be caused by dysfunction or damage to the nerve cells in the retina or macula.

[0212] NaIO3 (sodium iodate) has been reported to induce damage and / or degeneration in part or all of the outer and inner layers of the retina, and to induce damage to the retinal pigment epithelium (RPE) and photoreceptors (e.g., rod and cone cell layers) (AE-H. Koh et al., Journal of Photochemistry & Photobiology, B: Biology, 196(2019) 111514). Therefore, by using the NaIO3 model, preventative or therapeutic effects can be confirmed against diseases caused by nerve damage in the retina or macula, or against damage to the retinal pigment epithelium (RPE) and photoreceptors (e.g., rod and cone cell layers). Examples of such diseases include retinopathy, choroidal neovascularization, macular degeneration, retinal degeneration, macular edema, retinal swelling, color vision deficiency, retinal cell degeneration, retinal vascular occlusion, retinal detachment, hereditary retinal diseases, or any combination thereof.

[0213] As used herein, the term “retinal disease” refers to a disease or damage to the retina (e.g., the tissue lining the inner surface of the back of the eye that captures images through the cornea and lens) or retinal cells.

[0214] As used herein, the term “diabetic retinopathy” or “DR” refers to retinopathy caused by complications associated with diabetes. Depending on the severity of the disease, DR can be asymptomatic, may cause minor vision problems, or may lead to blindness. DR results from microvascular changes in the retina. Hyperglycemia-induced intramural pericyte death and basement membrane thickening can lead to vascular wall failure. These damages alter the formation of the blood-retinal barrier and make retinal vessels more permeable. Pericyte death can be induced when persistent hyperglycemia activates PCK-δ, encoded by protein kinase C-δ, and p38 mitogen-activated protein kinase (MAPK) and increases the expression of Src homology-2 domain-containing phosphatase-1 (SHP-1), a protein tyrosine phosphatase that is a previously unrecognized target of PKC-δ signaling. This signaling cascade leads to dephosphorylation of the PDGF receptor and a reduction in downstream signaling from this receptor, resulting in “pericytosis.” Small blood vessels, such as those in the eye, can be particularly vulnerable to poor blood sugar control. Excessive accumulation of glucose and / or fructose can damage small blood vessels in the retina.

[0215] DR can be divided into two distinct stages (Wu L. et al., World J Diabetes, 4(6): 290-294(2013)). The first stage is called nonproliferative diabetic retinopathy (NPDR), which is associated with early diabetic retinopathy. NPDR is usually asymptomatic or associated with mild visual distortion caused by fluid leakage into blood vessels in the surrounding tissues. The only way to detect NPDR is through fundus photography, which can show microaneurysms (tiny bulges in the walls of blood-filled arteries). If left untreated, DR patients can progress to a more advanced second stage called proliferative diabetic retinopathy (PDR). PDR is characterized by abnormal new blood vessel formation (i.e., neovascularization), which can rupture and bleed, resulting in blurred vision. Other symptoms of PDR include floaters or black lines in the visual field (floaters), visual changes, color vision deficiency, dark or empty areas in the visual field, pain, uneven vision, and complete vision loss.

[0216] The term "diabetic retinopathy" includes all types of diabetic retinopathy, including but not limited to nonproliferative diabetic retinopathy (NPDR), proliferative diabetic retinopathy (PDR), diabetic macular degeneration, and diabetic macular edema.

[0217] In some respects, PDR develops after the onset of NPDR (e.g., the subject is first diagnosed with NPDR and the disease progresses to PDR). In other respects, PDR develops independently of NPDR. As used herein, the term "diabetic retinopathy" also encompasses all types of diabetic retinopathy and any and all symptoms of diabetic retinopathy, regardless of cause. Non-limiting examples of risk factors for diabetic retinopathy include duration of diabetes, genetics, excessive alcohol consumption, smoking, hypertension, obesity, dyslipidemia, high cholesterol, kidney disease, pregnancy, and impaired kidney function.

[0218] As used herein, the term "macular degeneration" refers to any pathological condition of the macula (the central region of the retina associated with highly sensitive and accurate vision). In some respects, the terms "macular degeneration" and "retinopathy" may be used interchangeably (e.g., when only the macula is affected). In some respects, macular degeneration is diabetic macular degeneration.

[0219] Diabetic macular degeneration (AMD) occurs when the macula is affected by retinal changes caused by diabetes. The term encompasses two distinct eye conditions: diabetic macular edema and diabetic ischemic macular degeneration. The two types of macular degeneration are often comorbid; that is, people with macular edema usually also have ischemic macular degeneration. Ischemic macular degeneration occurs alongside macular edema and can occur even when macular edema is mild. In some respects, retinal changes associated with diabetic macular degeneration include decreased retinal potential within the retina, pericyte loss, cellless capillary formation, vascular congestion, vascular dysfunction, vascular leakage, vascular occlusion, tissue swelling (edema), tissue ischemia, or any combination thereof.

[0220] As used in this article, the term "cellless capillary" refers to a capillary-sized blood vessel that has no cell nucleus anywhere along its length.

[0221] As used herein, the term "vascular congestion" refers to a type of vascular injury that is an important factor in the pathogenesis of various ocular diseases disclosed herein, such as diabetic macular edema. Vascular congestion is associated with fluid accumulation (e.g., blood within the vascular system). In some respects, vascular congestion can be caused by hyperglycemia (i.e., hyperglycemia).

[0222] As used herein, the term “macular degeneration” refers to any of a variety of conditions and circumstances in which the central region of the retina (i.e., the macula) degenerates or loses its functional activity. Degeneration or loss of functional activity can occur due to, for example, cell death, reduced cell proliferation, loss of normal biological function, or any combination thereof. Macular degeneration can result in and / or manifest as alterations in the structural integrity of the macula’s cells and / or extracellular matrix, alterations in the composition of normal cells and / or extracellular matrix, and / or loss of function of the macula’s cells. Cells can be any cell type normally present in or near the macula, including RPE cells, photoreceptor cells (e.g., rod and cone cell layers), and / or capillary endothelial cells. Age-related macular degeneration is the most common form of macular degeneration, but the term “macular degeneration” does not necessarily exclude macular degeneration in non-aged patients. Non-limiting examples of macular degeneration include: age-related macular degeneration (wet or dry), Best macular dystrophy, Sorsby fundus dystrophy, familial dominant drusen (Malattia Leventinese), Doyne celluloid retinal dystrophy, Stargardt's disease (also known as Stargardt's macular dystrophy, juvenile macular degeneration, or fundus flavimaculatus), and macular degeneration associated with pigment epithelial detachment.

[0223] As used herein, the term "age-related macular degeneration" (AMD) generally refers to a retinal disease that affects older individuals and is associated with loss of central vision due to damage to the central region of the retina (i.e., the macula). AMD is typically characterized by the gradual accumulation or buildup of pale yellow, insoluble extracellular deposits called drusen (accumulation of extracellular proteins such as amyloid-β and lipids) within the macula (primarily between the retinal pigment epithelium (RPE) and the underlying choroid). This accumulation or buildup of deposits within the macula gradually damages it and leads to loss of central vision. As used herein, the term "macula" refers to the central region of the retina responsible for central high-resolution color vision.

[0224] Although several theories have been proposed, including oxidative stress, mitochondrial dysfunction, and inflammatory processes, the pathogenesis of age-related macular degeneration (AMD) is not fully understood. An imbalance between the production and degradation of damaged cellular components leads to, for example, the accumulation of harmful products such as intracellular lipofuscin and extracellular drusen. Early atrophy is characterized by thinning or depigmented areas of the retinal pigment epithelium (RPE) in early AMD, preceding geographic atrophy. In later stages of AMD, RPE atrophy (i.e., geographic atrophy) and / or the development of new blood vessels (i.e., neovascularization) lead to photoreceptor death and central vision loss. In dry (non-exudative) AMD, cellular debris called drusen accumulates between the retina and choroid, leading to retinal atrophy and scarring. In more severe wet (exudative) AMD, blood vessels grow from the choroid behind the retina (i.e., neovascularization) and exude exudates and fluid, which can also lead to hemorrhage.

[0225] Based on the extent of drusen present, AMD can be classified into three main stages: (i) early, (ii) intermediate, and (iii) advanced or late. Early AMD is characterized by the presence of multiple small (e.g., less than about 63 micrometers in diameter) drusen or a small number of medium-sized (e.g., about 63 to 124 micrometers in diameter) drusen. In the early stage, patients typically have no noticeable symptoms or vision loss. The intermediate stage is characterized by the presence of many medium-sized drusen or one or more large (e.g., greater than about 125 micrometers in diameter) drusen. At this stage, some patients may begin to experience blurred vision in the center of their field of vision. Advanced or late AMD is characterized by extensive damage to retinal tissue, leading to central scotoma and eventually central vision loss. Based on the type of damage (e.g., the presence or absence of neovascularization), advanced or late AMD can be further subdivided into two subtypes: (i) geographic atrophy (also known as atrophic AMD) and (ii) wet AMD (also known as neovascular or exudative AMD).

[0226] AMD has two main forms: (i) dry AMD and (ii) wet AMD. Unless otherwise stated, the term "age-related macular degeneration" includes both dry AMD and wet AMD. As used herein, the term "age-related macular degeneration" also encompasses all types of age-related macular degeneration and any and all symptoms of age-related macular degeneration, regardless of cause. Non-limiting examples of symptoms associated with macular degeneration (e.g., age-related macular degeneration) include: central vision loss, distortion, decreased contrast sensitivity, blurred vision, difficulty adapting to dim light, sudden onset and rapid deterioration of symptoms, and decreased color vision. In some respects, macular degeneration (e.g., age-related macular degeneration) can cause macular edema (e.g., swelling of the macular due to the accumulation of fluid and protein deposits on or under the macula).

[0227] As used in this article, the term "dry AMD" (also known as atrophic age-related macular degeneration or non-exudative AMD) refers to all forms of AMD that are not wet (neovascular) AMD. This includes early and intermediate forms of AMD as well as a late form of dry AMD called geographic atrophy. Patients with dry AMD often have mild symptoms in the early stages, while visual loss occurs more frequently as the disease progresses to geographic atrophy.

[0228] As used herein, the term "wet AMD" (also known as neovascular age-related macular degeneration or exudative AMD) refers to a retinal condition characterized by the presence of retinal neovascularization and represents the most advanced form of AMD. In wet AMD, vessels grow from choroidal capillaries and, in some cases, from the underlying retinal pigment epithelium through defects in Bruch's membrane (choroidal neovascularization or neovascularization). The serous or hemorrhagic exudate leaking from these vessels can lead to secondary degeneration of the neurosensory retina, detachment and rupture of the retinal pigment epithelium, vitreous hemorrhage, and the formation of fibrovascular scarring in the macular region, as well as permanent loss of central vision.

[0229] As used herein, the term “neovascularization” refers to the growth of new, abnormal blood vessels in different parts of the eye, which can lead to bleeding and vision loss. As used herein, the term “choroidal neovascularization” refers to the abnormal growth of new blood vessels in the choroid (i.e., the vascular layer of the eye containing connective tissue and located between the retina and sclera). In wet AMD, new blood vessels can grow into the retina through the retinal pigment epithelium (RPE) and choroid, impairing visual function due to leakage of blood and lipids. As used herein, the term “retinal neovascularization” refers to the abnormal development, proliferation, and / or growth of blood vessels on or within the retina (e.g., on the surface of the retina). Retinal neovascularization can occur in many retinopathy-associated with retinal ischemia (e.g., diabetic retinopathy, sickle cell retinopathy, retinal periphlebitis (Eales disease), ocular ischemia syndrome, carotid-cavernous fistula, familial exudative vitreoretinopathy, hyperviscosity syndrome, radiation retinopathy, retinal vein occlusion, retinal artery occlusion, retinal embolism, shotgun retinal choroidal disease, choroidal melanoma, chronic retinal detachment, anterior ischemic optic neuropathy (AION), non-arteritis anterior ischemic optic neuropathy (NAION), and pigmentary disorders). Methods for detecting neovascularization are known in the art, including but not limited to measuring the expression of CD31 (platelet endothelial cell adhesion molecule, also known as PECAM-1) and vascular endothelial growth factor (VEGF) in tissues; see, for example, Schluter A. et al., BMCCancer, 18(1):272 (2018).

[0230] As used in this article, the term "color vision deficiency" or "color vision impairment" refers to a condition in which color perception is impaired due to dysfunction of cone cells, a type of photoreceptor in the retina. Cone cells are photoreceptors, and dysfunction or loss of cone cells leads to color vision deficiency (Bennett J., Gene therapy for color blindness, N. Engl. J. Med., 2009, 361(25):2483-2484). In patients with AMD, reduced or lost cone cell function has also been reported to be accompanied by color vision deficiency in many cases (O'Neill-Biba M. et al., Loss of chromaticsensitivity in AMD and diabetes: a comparative study, Ophthalmic Physiol. Opt., 2010, 30(5):705-716). Models for assessing such color vision deficiencies can employ NaIO3 (sodium iodate), which induces oxidative stress in photoreceptors and causes cone cell apoptosis, thereby inducing color vision deficiencies (Wang J. et al., Direct effect of sodium iodate on neurosensory retina, Invest. Ophthalmol.Vis. Sci., 2014, 55(3):1941-1953; Takeda A. et al., New Insights Into Immunological Therapy for Retinal Disorders, Front. Immunol., 2020, 11:1431). In one embodiment, a polypeptide as described herein, comprising the TAFA amino acid sequence, fragments thereof, or variants thereof, protects cone cells and photoreceptors containing therefrom from NaIO3-induced oxidative stress, and this mechanism suggests that the polypeptide and the polynucleotide encoding it can be used as agents for the prevention or treatment of the aforementioned color vision deficiencies.

[0231] Color vision deficiencies can be divided into color blindness and color weakness. Color blindness refers to the inability to fully perceive one of the three primary colors, while color weakness refers to the ability to perceive colors but seeing them differently due to a deficiency in the corresponding receptors. Color vision deficiencies are common, occurring in approximately 5% to 8% of the male population. Among color vision deficiencies, deuteranomaly is usually the most common, accounting for about 25% to 45% of all cases. Following this, roughly in order, are deuteranopia (green color blindness), protanopia (red color blindness), and protanomaly (red color weakness), each occurring in about 1% of the male population. Trichromatic vision deficiencies and total color blindness are very rare, with a prevalence of approximately 0.005%.

[0232] As used herein, the term "hereditary retinal disease" refers to a retinal disease in which structural and functional abnormalities of retinal cells occur due to genetic defects or abnormalities. The onset time and symptoms of hereditary retinal diseases vary depending on the causative gene. Non-limiting examples of hereditary retinal diseases include retinitis pigmentosa (RP), Leber congenital amaurosis, Stargardt's disease, Coats retinopathy, cone dystrophy, choroidal agenesis, Usher syndrome, Best's disease, X-linked retinoschisis, hereditary color vision deficiency, and unidentified hereditary retinal dystrophy caused by unidentified genetic abnormalities.

[0233] As used in this article, the term "retinitis pigmentosa" (RP) refers to a retinal disease in which photoreceptor cells and retinal pigment epithelial cells are damaged (or degenerated). As photoreceptor cells are damaged, night blindness initially appears, followed by a gradual narrowing of the visual field, eventually leading to blindness. The primary cause is a defect in the genes involved in the mechanism of converting light into electrical signals within photoreceptor cells, and in some cases, genetic abnormalities are also found in retinal pigment cells. These genetic abnormalities cause widespread retinal damage.

[0234] In retinitis pigmentosa, the earliest clinically apparent feature is the gradual destruction of rod and cone cells, with rod cells being affected earlier than cone cells. When the destruction of photoreceptor cells progresses to the cone cells, central vision is lost, eventually leading to complete blindness.

[0235] As used herein, the term "Leber congenital amaurosis" refers to a hereditary retinal disorder that can cause congenital blindness at birth or shortly thereafter. In patients with Leber congenital amaurosis, the rod and cone cells of the normally functioning retina are absent, resulting in the absence of both cone and rod cell responses on electroretinography. Leber congenital amaurosis is a disease caused by a genetic abnormality, with mutations in the disease-causing genes found in approximately 40% to 50% of patients. Of the twelve known disease-causing genes, eleven (GUCY2D, RPE65, SPATA7, AIPL1, LCA5, RPGRIP1, CRB1, CEP290, IMPDH1, RD3, and RDH12) carry mutations that are inherited in an autosomal recessive manner, and, more rarely, mutations that are inherited in an autosomal dominant manner (CRX) have also been reported.

[0236] As used herein, the term “Stargardt’s disease” refers to a form of retinal dystrophy inherited in an autosomal recessive manner. Stargardt’s disease typically appears between the ages of 8 and 15, characterized by a progressive loss of central visual acuity due to bilateral macular degeneration. Mutations in a gene called ABCA4 are currently believed to be the cause of Stargardt’s disease. Mutations in ABCA4 lead to the accumulation of lipofuscin-like substances in the retinal pigment epithelium, resulting in RPE cell death and loss of photoreceptors (e.g., the rod and cone cell layers). Mutations in ABCA4 are associated with malnutrition of cone and rod cells, as well as severe forms of retinal dystrophy.

[0237] As used herein, the term "external exudative retinopathy" refers to a retinal vascular disease in which capillary dilation and aneurysmal dilation of retinal capillaries lead to the accumulation of exudates within and under the retina, causing exudative retinal detachment. It is known to be caused by a somatic mutation in the NDP gene on the X chromosome, which results in a deficiency of the norrin protein required for retinal development.

[0238] As used in this article, the term "cone dystrophy" refers to a disease caused by genetic abnormalities that lead to degeneration of the cone cells in the retina responsible for color vision and central vision, resulting in loss of central vision. Cone dystrophy is broadly classified into pure cone dystrophy and cone-rod dystrophy. The inheritance patterns are diverse, including autosomal dominant, autosomal recessive, and X-linked patterns, and due to de novo mutations, the disease can also occur without a clear familial pattern.

[0239] As used in this article, the term "achoroidemia" refers to a rare, X-linked, progressive degeneration of the choroid, retinal pigment epithelium, and photoreceptors. In affected patients, mutations in the CHM gene lead to a deficiency of Rab escort protein 1 (REP1), causing photoreceptors in the retina to lose function and gradually die. Symptoms typically occur in men, usually beginning with night blindness in childhood, followed by progressive loss of peripheral vision, while central vision is relatively preserved in the early stages. Many affected men maintain good visual acuity but have a significantly narrowed visual field in their 40s, and visual acuity is also lost between approximately 50 and 70 years of age. Some patients also exhibit impaired color perception.

[0240] As used in this article, the term "Usher syndrome" refers to a hereditary condition in which visual impairment progresses in association with hearing loss. Hearing loss in Usher syndrome is due to an inner ear abnormality, while the visual impairment is associated with retinitis pigmentosa (RP).

[0241] As used herein, the term “Best’s disease” refers to a hereditary retinal disorder caused by mutations in the BEST1 (VMD2) gene. This disease progresses slowly and can lead to decreased central visual acuity. Mutations in the BEST1 (VMD2) gene impair the function of bestrophin (a calcium-activated chloride channel protein in the basolateral membrane of the retinal pigment epithelium), thereby disrupting fluid transport across the retinal pigment epithelium and leading to serous retinal detachment and / or retinal pigment epithelium detachment.

[0242] As used in this article, the term "X-linked retinoschisis" refers to a condition in which the inner layers of the retina are split due to a mutation in the retinoschisis gene (RS1). Retinoschisis is characterized by abnormal separation of the retinal layers, particularly the nerve fiber layers, among the ten layers that make up the retina, leading to visual impairment. Juvenile X-linked retinoschisis is a rare X-linked recessive genetic disorder with a global prevalence of approximately 1 in 120,000.

[0243] As used herein, the term "neuralgic pain" refers to pain caused by damage, impairment, and / or dysfunction affecting the central nervous system (CNS) and / or peripheral nervous system at any level. As used herein, the term "neuralgic pain" encompasses any and all types of neuropathic pain, regardless of its cause or the full range of its symptoms.

[0244] Neuropathic pain includes central neuropathic pain and peripheral neuropathic pain. As used herein, the term "central neuropathic pain" refers to pain caused by a disease, congenital defect, or injury to the central nervous system (i.e., the brain or spinal cord). As used herein, the term "peripheral neuropathic pain" refers to pain caused by damage or infection to peripheral sensory nerves.

[0245] Symptoms of neuropathic pain may include persistent or chronic pain, spontaneous pain and atypical pain (e.g., a painful response to a normally painless stimulus), hyperalgesia (e.g., a significant painful response to a stimulus that is usually only slightly unpleasant, such as a pinprick), hyperesthesia (e.g., excessive somatic sensitivity to stimuli, particularly skin stimuli), or hyperalgesia (e.g., when a brief unpleasant sensation becomes prolonged, severe pain). In some implementations, symptoms may be persistent and may persist even after the primary underlying cause (if present) has subsided. See, for example, Merck Manual, Neuropathic Pain, merckmanuals.com / professional / neurologic-disorders / pain / neuropathic-pain; Campbell JN and Meyer RA, Neuron 52(1):77-92 (2006).

[0246] In some implementations, the types of neuropathic pain include (1) neuralgia, (2) deafferent neuralgia syndrome, (3) complex regional pain syndrome (CRPS), and (4) neuropathy (central or peripheral).

[0247] In some implementations, neuropathic pain is defined as pain radiating along one or more specific nerves (e.g., cranial nerves) without any apparent pathological changes in the neurostructure. Neuropathic pain includes, but is not limited to, trigeminal neuralgia (TN), atypical trigeminal neuralgia (ATN), occipital neuralgia, glossopharyngeal neuralgia, postherpetic neuralgia (caused by shingles or herpes virus infection), pain due to peripheral nerve injury, sciatica, low back pain, and atypical facial pain. Chemical irritants, chronic kidney disease, diabetes, inflammation, trauma (including surgery), nerve compression from adjacent structures (e.g., tumors), certain medications (e.g., cisplatin, paclitaxel, or vincristine), porphyria (blood disorders), and infections (e.g., herpes zoster, HIV / AIDS, Lyme disease, or syphilis) can all cause neuropathic pain.

[0248] In some implementations, neuropathic pain is a deafferent neuralgia syndrome, which can be caused by loss of sensory input from parts of the body (e.g., by interruption of peripheral sensory fibers or nerves within the central nervous system). Deafferent neuralgia syndromes include, but are not limited to, brain or spinal cord injury, post-stroke pain, phantom limb pain, paraplegia, brachial plexus avulsion, and lumbar radiculopathies.

[0249] In some implementations, neuropathic pain is complex regional pain syndrome (CRPS), a chronic pain condition that most commonly affects the arm or leg. In some implementations, CRPS occurs after trauma, surgery, stroke, or myocardial infarction. In some implementations, CRPS is CRPS type I (CRPS-I) (also known as reflex sympathetic dystrophy syndrome), in which the affected individual typically does not have a diagnosed nerve injury. In other implementations, CRPS is CRPS type II (CRPS-II) (also known as burning neuropathic pain), which is associated with a diagnosed nerve injury.

[0250] In some embodiments, neuropathic pain is a neuropathy, which refers to pain caused by functional or pathological changes in nerves (e.g., due to disease or injury). Neuropathies are typically clinically characterized by abnormalities in sensory and / or motor neurons. In some embodiments, a neuropathy is a central nervous system neuropathy (e.g., functional or pathological changes in the central nervous system). In other embodiments, a neuropathy is a peripheral neuropathy (e.g., functional or pathological changes affecting one or more peripheral nerves (including motor nerves, sensory nerves, autonomic nerves, or combinations thereof). In some embodiments, a peripheral neuropathy involves functional or pathological changes affecting a single nerve or group of nerves (i.e., mononeuropathy). In some embodiments, a peripheral neuropathy involves functional or pathological changes affecting multiple nerves (local or systemic) (i.e., polyneuropathy). In some embodiments, a peripheral neuropathy affects both sides of the body in a nearly symmetrical manner (i.e., symmetrical polyneuropathy). In some embodiments, a peripheral neuropathy affects anatomically heterogeneous areas of the body (e.g., polymononeuritis or multifocal mononeuropathy, polymononeuropathy).

[0251] As used herein, the term "mononeuropathy" refers to a peripheral neuropathy characterized by loss of motor function and / or sensation in the area innervated by a single peripheral nerve or nerve group due to injury or destruction of that single peripheral nerve or nerve group. Mononeuropathy is typically caused by localized injury or trauma resulting in persistent compression or pressure on the single nerve. Certain systemic conditions (e.g., polyneuropathy) can also cause mononeuropathy. In some embodiments, the localized injury or trauma results in the destruction of all or part of the myelin sheath covering the nerve or neuronal axon, thereby slowing or blocking impulse conduction along the nerve. Mononeuropathy can affect any part of the body. Non-limiting examples of mononeuropathy-related pain include sciatic nerve dysfunction, generalized pelvic nerve dysfunction, radial nerve dysfunction, ulnar nerve dysfunction, cranial nerve VI mononeuropathy, cranial nerve VII mononeuropathy, cranial nerve III mononeuropathy (compression type), cranial nerve III mononeuropathy (diabetic type), axillary nerve dysfunction, carpal tunnel syndrome, femoral nerve dysfunction, tibial nerve dysfunction, Bell's palsy, thoracic outlet syndrome, and sixth (abduced) nerve palsy (see, for example, Finnerup NB et al., Pain 157(8):1599-1606 (2016); National Institute of Neurological Disorders and Stroke, Peripheral Neuropathy Fact Sheet, ninds.nih.gov / disorders / peripheralneuropathy / detailj3eripheralneuropathy.htm). In some implementations, mononeuropathy pain is sciatica.

[0252] As used herein, the term "polyneuropathy" refers to a peripheral neuropathy characterized by loss of motor function and / or sensation in the area innervated by multiple peripheral nerves due to injury or destruction of multiple peripheral nerves. Polyneuropathy pain includes, but is not limited to, pain associated with post-poliomyelitis syndrome, post-mastectomy pain syndrome, diabetic neuropathy, alcoholic neuropathy, amyloidosis, toxin-induced neuropathy, AIDS-related neuropathy, hypothyroid neuropathy, uremic neuropathy, vitamin deficiency-related neuropathy, chemotherapy-induced pain, neuropathy associated with 2',3'-dideoxycytidine (ddC) treatment, Guillain-Barré syndrome, and Fabry disease (see, for example, Finnerup NB et al., Pain 157(8):1599-1606 (2016); National Institute of Neurological Disorders and Stroke, Peripheral Neuropathy FactSheet, ninds.nih.gov / disorders / peripheralneuropathy / detail3eripheralneuropathy.htm). In some implementations, polyneuropathy is diabetic peripheral neuropathy, which can be caused by elevated blood glucose levels (hyperglycemia) and / or elevated blood lipid levels (e.g., triglycerides) in subjects with diabetes, thereby inducing peripheral nerve damage in the subjects.

[0253] In some embodiments, the peripheral neuropathy described herein is classified according to the damaged or affected neuronal portion (e.g., axon, myelin sheath, or cell body). In some embodiments, peripheral neuropathy is distal axonopathy, which is caused by metabolic and / or toxic disorders of the axon. Metabolic disorders may include deficiency syndromes such as diabetes, renal failure, malnutrition, and alcoholism. In some embodiments, the metabolic disorder is diabetes, and the distal axonopathy is diabetic neuropathy.

[0254] In other embodiments, peripheral neuropathy is myelinating neuropathy, which is caused by primary damage to the myelin sheath and / or Schwann cells that form the myelin sheath, leading to acute failure of impulse conduction. The most common cause of myelinating neuropathy is acute inflammatory demyelinating polyneuropathy (AIDP; also known as Guillain-Barré syndrome), but other causes include chronic inflammatory demyelinating polyneuropathy (CIDP), inherited metabolic disorders (e.g., leukodystrophy), and toxins.

[0255] In other embodiments, peripheral neuropathy is a neuropathy caused by the destruction of neurons in the peripheral nervous system (PNS). Neuropathies can be caused by, for example, motor neuron disease, sensory neuron disease (e.g., caused by herpes zoster), toxins, or autonomic dysfunction, and can also be caused by exposure to neurotoxic chemotherapeutic agents such as vincristine.

[0256] Neuropathic pain can be caused or associated with a variety of etiologies, including but not limited to physical injury (e.g., trauma or repetitive stress), disease or condition, exposure to toxic agents, or any combination thereof. In some embodiments, neuropathic pain is caused or associated with traumatic injury or damage, such as nerve compression injury (e.g., nerve squeeze, nerve stretch, nerve entrapment, or incomplete nerve transection), spinal cord injury (e.g., hemisection), injury or damage to peripheral nerves (e.g., motor, sensory, and / or autonomic nerves), limb amputation, contusion, inflammation (e.g., myelitis), or surgery. In some embodiments, neuropathic pain is caused or associated with repetitive stress involving repetitive, slow, and / or forceful activities requiring prolonged movement of any joint group. Unbound by theory, such repetitive stress can lead to inflammation and swelling of ligaments, tendons, and muscles, thereby narrowing the anatomical passage through which nerves pass (e.g., ulnar neuropathy and carpal tunnel syndrome, where a nerve at the elbow or wrist is entrapped or compressed). In other embodiments, the neuropathic pain is caused by or associated with a disease or condition, including but not limited to ischemic events (e.g., stroke or myocardial infarction), multiple sclerosis, metabolic and / or endocrine diseases or conditions (e.g., diabetes, other metabolic diseases, and acromegaly caused by excessive production of growth hormone, characterized by abnormal enlargement of skeletal elements (including joints), leading to nerve compression and pain), small vessel diseases that reduce oxygen supply to peripheral nerves and cause damage to nerve tissue (e.g., vasculitis, i.e., vascular inflammation), and autoimmune diseases (e.g., Sjögren's syndrome). Syndrome, lupus, rheumatoid arthritis, and acute inflammatory demyelinating polyneuropathy, also known as Guillain-Barré syndrome; kidney disease; cancer or tumors (e.g., neoplastic tumors, neuromas, paraneoplastic syndromes, and toxicity caused by chemotherapy agents and / or radiation used in cancer treatment); infections (e.g., herpes zoster, Epstein-Barr virus, West Nile virus, cytomegalovirus, herpes simplex virus, acquired immunodeficiency syndrome (AIDS), Lyme disease, diphtheria, and leprosy); inflammatory conditions; peripheral neuropathy (e.g., neuroma); and hereditary or de novo hereditary conditions (e.g., Charcot-Marie-Tooth disease, characterized by weakness and atrophy of the muscles in the legs and feet, gait abnormalities, loss of tendon reflexes, and lower limb paralysis); and mononeuropathy or polyneuropathy. In some embodiments, the neuropathic pain is caused by or associated with a pathogen infection, including, for example, tick-borne infection, varicella-zoster virus, Epstein-Barr virus, West Nile virus, cytomegalovirus, herpes simplex virus, or AIDS.In other embodiments, neuropathic pain is caused by or related to exposure to toxic agents such as drugs, alcohol, heavy metals (e.g., lead, arsenic, or mercury), industrial reagents (e.g., fumes from solvents or adhesives), or nitrous oxide.

[0257] As used herein, the expression “neuralgia associated with a disease or condition” means neurogia that is associated with, caused by, or resulting from a disease or condition (e.g., any disease or condition described herein).

[0258] As used herein, the terms “nucleic acid,” “nucleic acid molecule,” “nucleotide sequence,” “polynucleotide,” and their variants are used interchangeably and refer to a nucleotide sequence linked by phosphodiester bonds. Polynucleotides are indicated herein in 5' to 3' orientations. The polynucleotides disclosed herein can be deoxyribonucleic acid (DNA) molecules or ribonucleic acid (RNA) molecules. Nucleotide bases are indicated herein by single-letter codes such as A (adenine), G (guanine), T (thymine), C (cytosine), I (inosine), and U (uracil).

[0259] As used herein, the terms “operably linked” and “operably associated” refer to the arrangement of DNA sequences such that these sequences are in a functional relationship and cooperate to perform a desired function. For example, a given promoter can be operably linked to a coding sequence (e.g., transgenes) when the promoter is positioned to drive transcription of the coding sequence. The promoter and coding sequence do not need to be adjacent as long as the functional relationship is maintained.

[0260] As used herein, the terms “pharmaceuticalally acceptable carrier” and “pharmaceuticalally acceptable excipient” and variations thereof refer to any carrier or diluent approved by a U.S. federal regulatory agency or listed in the United States Pharmacopeia for use in animals (including humans), or otherwise pharmaceutically acceptable in the subject, and which will not diminish the biological activity or properties of the administered compound to such an extent that administration of the composition becomes inappropriate or causes an undesirable physiological effect that would prevent administration to the subject. Pharmaceutically acceptable carriers and excipients used to prepare pharmaceutical compositions are generally safe, non-toxic, and suitable for administration in the context described herein.

[0261] As used herein, the term "pharmaceutical composition" refers to a composition comprising a mixture, suspension, or combination of one or more substances described herein (e.g., polypeptides, polynucleotides, carriers, cells, and / or recombinant viruses) and one or more additional chemical components (e.g., pharmaceutically acceptable carriers or excipients).

[0262] As used herein, the terms "promoter" and "promoter sequence" are used interchangeably and refer to a DNA sequence capable of regulating the expression of a coding sequence or functional RNA. Typically, the coding sequence is located at the 3' end of the promoter sequence. Promoters can be entirely derived from natural genes, may contain different elements derived from naturally occurring different promoters, and / or may also contain synthetic DNA segments. Those skilled in the art will understand that different promoters can direct gene expression in different tissues or cell types, at different developmental stages, or in response to different environmental or physiological conditions. Promoters that direct gene expression in common or most host cell types are generally referred to as "constitutive promoters." Promoters that direct gene expression in specific cell types are generally referred to as "cell-specific promoters" or "tissue-specific promoters." Promoters that direct gene expression at specific developmental stages or cell differentiation stages are generally referred to as "development-specific promoters" or "differentiation-specific promoters." Promoters that are activated and induce gene expression when cells are exposed to inducers, biomolecules, chemicals, ligands, light, etc., are generally referred to as "inducible promoters" or "regulatory promoters." In many cases, DNA fragments of different lengths can exhibit essentially the same promoter activity because the precise boundaries of the regulatory sequence are not fully defined.

[0263] Promoter sequences typically adjoin the transcription start site at their 3' end and extend upstream (in the 5' direction) sufficiently to include the minimum number of nucleotides or elements required to initiate transcription at detectable levels above background. Within the promoter sequence, the protein-binding domain (a common sequence) responsible for binding RNA polymerase, and the transcription start site (e.g., conveniently defined by S1 nuclease localization), can be found. In some embodiments, promoters suitable for use in this disclosure include tissue-specific promoters.

[0264] As used herein, the terms "gene regulatory region" and "regulatory region" refer to nucleotide sequences located upstream (5' untranslated sequence), inside, or downstream (3' untranslated sequence) of a coding region that influence transcription, RNA processing, RNA stability, or translation of the relevant coding region. Regulatory regions may include, for example, promoters, pretranslational sequences, introns, polyadenylation recognition sequences, RNA processing sites, effector binding sites, or stem-loop structures. When a coding region is intended for expression in eukaryotic cells, polyadenylation signals and transcription termination sequences are typically located at the 3' end of the coding sequence.

[0265] In some aspects, the polynucleotides described herein (e.g., transgenic polynucleotides comprising a polypeptide encoding an amino acid sequence of a TAFA protein, a fragment thereof, or a variant thereof, and a non-translated nucleic acid sequence) also comprise a promoter operatively associated with one or more coding regions and / or one or more additional expression (e.g., transcription) regulatory elements. In operative association, the coding region for a gene product is linked to one or more regulatory regions in such a way that the expression of the gene product is under the control or influence of one or more regulatory regions. For example, the coding region and the promoter are operatively associated, wherein activation of the promoter function results in transcription of the mRNA encoding the gene product specified by the coding region, and wherein 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. In addition to promoters, other expression regulatory elements such as enhancers, operons, repressors, and transcription termination signals may also be operatively associated with the coding region that directs the expression of the gene product.

[0266] As used herein, the terms “object,” “patient,” “individual,” and “host,” and variations thereof, are used interchangeably and refer to any mammalian object to which any of the compositions described herein (e.g., peptides, polynucleotides, recombinant expression constructs, vectors, cells, pharmaceutical compositions, or recombinant viruses) are administered. Non-limiting examples include humans, companion animals (e.g., dogs and cats), farm animals (e.g., cattle, sheep, pigs, and horses), and laboratory animals (e.g., non-human primates, rats, mice, rabbits, and guinea pigs). In a preferred embodiment, the object is a human. The methods described herein are suitable for human therapeutic or preventative use as well as veterinary use.

[0267] As used herein, the phrase “objects in need” includes objects to which the application of the compositions described herein would be beneficial, such as mammalian objects.

[0268] As used herein, the term "therapeutic effective amount" refers to an amount of a reagent or pharmaceutical composition comprising a composition of this disclosure (e.g., a polypeptide comprising a TAFA protein fragment or a variant thereof, or a polynucleotide encoding thereof) sufficient to achieve the desired therapeutic, pharmacological, and / or physiological effect in a subject of need. Because prevention can be considered a form of treatment, a therapeutic effective amount can also be a "preventive effective amount."

[0269] As used herein, the term "transgenic" refers to a polynucleotide encoded by a recombinant expression construct, including at least one polynucleotide (e.g., a polynucleotide encoding a TAFA protein, or a polynucleotide encoding a polypeptide containing a fragment of the TAFA protein or a variant thereof), a polynucleotide region, an expression product of such polynucleotide or region, a polynucleotide encoding a polypeptide or multi-peptide product, or a promoting or regulating nucleic acid. In some respects, the transgenic is heterologous to the cells in which it is introduced (or transduced) (e.g., it is not naturally expressed in the cells).

[0270] As used herein, the terms “treatment,” “management,” and “therapeutic” refer to any intervention that, for example, reduces the severity of a disease or condition, shortens the course of a disease, improves or eliminates one or more symptoms associated with a disease or condition, or provides a beneficial effect to a person suffering from such a disease or condition, even if it does not necessarily cure the disease or condition. The term also covers the prevention or suppression of the onset of a disease or condition or its symptoms.

[0271] As used herein, the terms “vector” and “construct” refer to any vector into which a nucleic acid or gene can be inserted and which is capable of delivering a nucleic acid sequence into a cell, wherein the nucleic acid sequence can be replicated and / or expressed. The nucleic acid sequence inserted into the vector can be exogenous or heterologous and can be transgenic. Examples of constructs include, but are not limited to, plasmids, granules, and viruses (e.g., AAV). Those skilled in the art can construct such vectors or constructs using standard recombination techniques (see, for example, Maniatis et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, NY, 1988; Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, Inc., NY, 1994). As used herein, the terms “expression vector” and “expression construct” refer to a vector or construct containing at least a portion of a nucleotide sequence encoding a gene product, which is transcribed, and in some cases, the resulting RNA is translated into a protein, polypeptide, or peptide. Expression constructs may include various regulatory elements, and in addition to regulatory sequences controlling transcription and translation, vectors and expression vectors may also contain nucleotide sequences that provide additional functionality. Viruses suitable for use in this invention include, but are not limited to, retroviruses, herpes simplex viruses, lentiviruses, poxviruses, vaccinia viruses, rhabdoviruses, adenoviruses, helper-dependent adenoviruses, and adeno-associated viruses (AAVs).

[0272] Vectors can be engineered to encode selection markers or reporter substances, allowing for the selection or identification of cells containing the vector. Expression of the selection marker or reporter substance enables the identification and / or selection of host cells that have integrated and expressed additional coding regions present in the vector. Non-limiting examples of selection marker genes known and used in the art include genes conferring resistance to ampicillin, streptomycin, gentamicin, kanamycin, hygromycin, the herbicide diprofenofos, or sulfonamides, as well as genes used as phenotypic markers, such as anthocyanin regulatory genes and isopentenyltransferase genes. Non-limiting examples of reporter substances known and used in the art include luciferase (Luc), green fluorescent protein (GFP), chloramphenicol acetyltransferase (CAT), β-galactosidase (LacZ), and β-glucuronidase (Gus). Selection markers can also be used as reporter substances.

[0273] Typically, recombinant adeno-associated virus (AAV) is produced by transfecting host cells (e.g., HEK293 cells) with three plasmids. These three plasmids are: (1) an AAV transfer plasmid containing gene expression components flanked by inverted terminal repeats (ITRs); (2) a “Rep-Cap plasmid” providing the Rep protein required for AAV genome replication and the capsid protein for viral particle formation; and (3) a “helper plasmid” containing adenoviral proteins (E2a, E4) and RNA (VA RNA) supporting the AAV life cycle. AAV particles are produced when these three types of plasmids are co-transfected into HEK293 cells or other cells that provide the function of the adenoviral E1 and E3 genes.

[0274] As used herein, the term "dual helper plasmid" refers to two or more required plasmids that provide AAV production in a cell. It will be apparent from this disclosure that, in some respects, the dual helper plasmids described herein provide both the required (2) Rep-Cap function and the required (3) auxiliary function. For example, in some respects, the dual helper plasmid comprises the rep gene, cap gene, E2a gene, E4 gene, and VA RNA gene.

[0275] The dual helper plasmids described herein not only contain the aforementioned genes but also arrange these genes in a specific conformation within the plasmid. For example, in some aspects, the E2a gene, E4 gene, and VA RNA gene are sequentially linked within the dual helper plasmid, and the rep gene and cap gene (collectively referred to herein as "rep-cap genes") are sequentially linked clockwise (5' to 3') between the 5' end of the E2a gene and the 3' end of the VA RNA gene. More specifically, in some aspects, the 5' end of the rep-cap gene is linked to the 5' end of the E2a gene, and the 3' end of the rep-cap gene is linked to the 3' end of the VA RNA gene. In other aspects, the E2a gene, E4 gene, and VA RNA gene are sequentially linked, and the rep-cap gene is located counterclockwise (3' to 5') between the 5' end of the E2a gene and the 3' end of the VA RNA gene. More specifically, in some respects, the 3' end of the rep-cap gene is linked to the 5' end of the E2a gene, and the 5' end of the rep-cap gene is linked to the 3' end of the VA RNA gene.

[0276] As used herein, the term "cell" includes both eukaryotic and prokaryotic cells and refers to any transformable cell capable of replicating a vector and / or expressing a gene encoded by the vector. Cells can be transfected, transduced, or transformed using vectors, which, as used herein, refers to the process of delivering or introducing exogenous polynucleotides (nucleic acid molecules) into a host cell. As used herein, the term "transformation," in its broadest sense, encompasses both transfection and transduction.

[0277] The host cell of the present invention is not particularly limited, but is preferably insect cell or mammalian cell, more preferably insect cell such as Sf9 cell, and mammalian cell such as HEK293 cell, HeLa cell, ARPE-19 cell, RPE-1 cell, HepG2 cell, Hep3B cell, Huh-7 cell, C8D1a cell, Neuro2A cell, CHO cell, MES13 cell, BHK-21 cell, COS7 cell, COP5 cell, A549 cell, MCF-7 cell, HC70 cell, HCC1428 cell, BT-549 cell, PC3 cell, LNCaP cell, Capan-1 cell, Panc-1 cell, MIA cell, etc. PaCa-2 cells, SW480 cells, HCT166 cells, LoVo cells, A172 cells, MKN-45 cells, MKN-74 cells, Kato-III cells, NCI-N87 cells, HT-144 cells, SK-MEL-2 cells, SH-SY5Y cells, C6 cells, HT-22 cells, PC-12 cells, or NIH3T3 cells. In some respects, the host cells are isolated host cells.

[0278] II. Polypeptides

[0279] In one aspect, this disclosure provides polypeptides having the ability to increase neurite length and / or the number of branching points.

[0280] In a preferred embodiment, the polypeptide comprises the amino acid sequence of a TAFA (TAFA chemokine-like family member) protein, or a fragment or variant thereof. In a preferred embodiment, the polypeptide comprises the amino acid sequence of general formula 7 from the N-terminus to the C-terminus:

[0281] General Formula 7:

[0282]

[0283] In general formula 1:

[0284] X1 does not exist, or is V, I, or L;

[0285] X2 is K, E, R, or Q;

[0286] X3 can be G, T, Q, P, or A;

[0287] X4 is either V or I;

[0288] X5 can be A, L, V, or I;

[0289] X6 is either R or L;

[0290] X7 is K, R, or Q;

[0291] X8 is either R or K;

[0292] X9 is either R or L;

[0293] X10 is either V or G;

[0294] X11 is either K or N;

[0295] X12 is either F or L;

[0296] X13 is either P or S;

[0297] X14 is either Q or K;

[0298] X15 is R, H, or Q;

[0299] X16 can be A, N, S, or T;

[0300] X17 is A, Q, R, K, or T;

[0301] X18 is either D or E;

[0302] X19 is either S or A;

[0303] X20 can be I, E, L, A, or V;

[0304] X21 is Q, G, or E;

[0305] X22 is either K or R;

[0306] X23 is H, Q, or E;

[0307] X24 can be E, Q, N, D, S, or H;

[0308] X25 is available in L, V, or M sizes;

[0309] X26 can be E, D, P, L, or A;

[0310] X27 is either E or D;

[0311] X28 can be V, T, A, or I;

[0312] X29 can be L, N, R, Y, S, or Q;

[0313] X30 is available in S, K, or T;

[0314] X31 is either S or M;

[0315] X32 can be S, A, or Y;

[0316] X33 can be S, T, or R;

[0317] X34 is either N or H;

[0318] X35 is either V or I;

[0319] X36 is either R or K;

[0320] X37 does not exist, or is V, A, G, M, or N;

[0321] X38 does not exist, or is T, I, N, F, or S; and

[0322] X39 does not exist, or is R, H, V, K, I, or Q.

[0323] In a preferred embodiment, the polypeptide comprises one or more amino acid sequences selected from the amino acid sequences shown in SEQ ID NO: 87 to SEQ ID NO: 141.

[0324] In a preferred embodiment, the polypeptide may consist of 8 to 61 amino acid residues.

[0325] In some embodiments, the polypeptide may consist of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, or 61 amino acid residues.

[0326] In a preferred embodiment, the polypeptide comprises an amino acid sequence having at least 50% sequence identity with the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 15 or SEQ ID NO: 87.

[0327] In some embodiments, the polypeptide has at least 50%, at least 51%, at least 52%, at least 53%, at least 54%, at least 55%, at least 56%, at least 57%, at least 58%, at least 59%, at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, 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 of SEQ ID NO: 1, SEQ ID NO: 15, or SEQ ID NO: 87.

[0328] In a preferred embodiment, the amino acid sequence of the polypeptide, from the N-terminus to the C-terminus, comprises the amino acid sequence of general formula 1:

[0329] General Formula 1:

[0330]

[0331] In general formula 1,

[0332] X1 can be E, D, P, L, or A;

[0333] X2 is either D or E; and

[0334] X3 can be T, V, I, or A.

[0335] In some embodiments, the polypeptide comprising the amino acid sequence of formula 1 comprises the amino acid sequence of SEQ ID NO: 142.

[0336] In some embodiments, the polypeptide comprising the amino acid sequence of formula 1 comprises an amino acid sequence selected from SEQ ID NO: 1 to SEQ ID NO: 14.

[0337] In some embodiments, the polypeptide comprising the amino acid sequence of general formula 1 may consist of 8 to 43 amino acid residues.

[0338] In some embodiments, such polypeptides may consist of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, or 43 amino acid residues.

[0339] In a preferred embodiment, the amino acid sequence of the polypeptide, from the N-terminus to the C-terminus, comprises the amino acid sequence of general formula 2:

[0340] General Formula 2:

[0341]

[0342] In general formula 2,

[0343] X1 can be E, D, P, L, or A;

[0344] X2 is either D or E;

[0345] X3 can be T, V, I, or A;

[0346] X4 can be I, E, A, L, or V;

[0347] X5 is Q, E, or G;

[0348] X6 is either K or R;

[0349] X7 is E, H, or Q;

[0350] X8 can be E, Q, N, D, S, or H; and

[0351] X9 can be L, M, or V.

[0352] In some embodiments, a polypeptide comprising the amino acid sequence of general formula 2 may comprise the amino acid sequence of SEQ ID NO: 143.

[0353] In some embodiments, a polypeptide comprising the amino acid sequence of formula 2 may comprise an amino acid sequence selected from SEQ ID NO: 28 to SEQ ID NO: 51.

[0354] In a preferred embodiment, the amino acid sequence of the polypeptide, from the N-terminus to the C-terminus, comprises the amino acid sequence of general formula 3:

[0355] General Formula 3:

[0356]

[0357] In general formula 3,

[0358] X1 can be E, D, P, L, or A;

[0359] X2 is either D or E;

[0360] X3 can be T, V, I, or A;

[0361] X4 can be Y, S, or L;

[0362] X5 is either S or T;

[0363] X6 is either S or T; and

[0364] X7 is either V or I.

[0365] In some embodiments, a polypeptide comprising the amino acid sequence of formula 3 may comprise the amino acid sequence of SEQ ID NO: 144.

[0366] In some embodiments, a polypeptide comprising the amino acid sequence of formula 3 may comprise an amino acid sequence selected from SEQ ID NO: 52 to SEQ ID NO: 58.

[0367] In some embodiments, the amino acid sequence of the polypeptide may include one or more amino acid sequences selected from SEQ ID NO: 152 to SEQ ID NO: 171.

[0368] In a preferred embodiment, the amino acid sequence of the polypeptide, from the N-terminus to the C-terminus, comprises the amino acid sequence of general formula 4:

[0369] Formula 4:

[0370]

[0371] In general formula 4,

[0372] X1 is either Q or K;

[0373] X2 is R, H, or Q;

[0374] X3 can be A, N, S, or T;

[0375] X4 can be R, A, Q, K, or T;

[0376] X5 is either D or E;

[0377] X6 is either A or does not exist; and

[0378] X7 is S, A, or does not exist.

[0379] In some embodiments, a polypeptide comprising the amino acid sequence of formula 4 may comprise the amino acid sequence of SEQ ID NO: 145.

[0380] In some embodiments, a polypeptide comprising the amino acid sequence of formula 4 may comprise an amino acid sequence selected from SEQ ID NO: 15 to SEQ ID NO: 27.

[0381] In some embodiments, the polypeptide containing the amino acid sequence of general formula 4 may consist of 15 to 46 amino acid residues.

[0382] In some embodiments, such polypeptides may consist of 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, or 46 amino acid residues.

[0383] In a preferred embodiment, the amino acid sequence of the polypeptide, from the N-terminus to the C-terminus, comprises the amino acid sequence of general formula 5:

[0384] Formula 5:

[0385]

[0386] In general formula 5,

[0387] X1 is either Q or K;

[0388] X2 is R, H, or Q;

[0389] X3 can be A, N, S, or T;

[0390] X4 can be R, A, Q, K, or T;

[0391] X5 is either D or E;

[0392] X6 is either A or does not exist;

[0393] X7 is S, A, or does not exist;

[0394] X8 is either R or K;

[0395] X9 is either R or L;

[0396] X10 is either V or G;

[0397] X11 is either K or N;

[0398] X12 is either F or L; and

[0399] X13 is either P or S.

[0400] In some embodiments, a polypeptide comprising the amino acid sequence of formula 5 may comprise the amino acid sequence of SEQ ID NO: 146.

[0401] In some embodiments, a polypeptide comprising the amino acid sequence of formula 5 may comprise an amino acid sequence selected from SEQ ID NO: 59 to SEQ ID NO: 74.

[0402] In a preferred embodiment, the amino acid sequence of the polypeptide, from the N-terminus to the C-terminus, comprises the amino acid sequence of general formula 6:

[0403] Formula 6:

[0404]

[0405] In general formula 6,

[0406] X1 is either Q or K;

[0407] X2 is R, H, or Q;

[0408] X3 can be A, N, S, or T;

[0409] X4 can be R, A, Q, K, or T;

[0410] X5 is either D or E;

[0411] X6 is either A or does not exist;

[0412] X7 is S, A, or does not exist;

[0413] X8 can be I, A, V, or L;

[0414] X9 is either Q or E;

[0415] X10 is either H or Q;

[0416] X11 is N, D, S, or H; and

[0417] X12 is either L or M.

[0418] In some embodiments, a polypeptide comprising the amino acid sequence of formula 6 may comprise the amino acid sequence of SEQ ID NO: 147.

[0419] In some embodiments, a polypeptide comprising the amino acid sequence of formula 6 may comprise an amino acid sequence selected from SEQ ID NO: 75 to SEQ ID NO: 85.

[0420] In some embodiments, the amino acid sequence of the polypeptide may include one or more amino acid sequences selected from SEQ ID NO: 172 to SEQ ID NO: 184.

[0421] III. Nucleic Acids

[0422] On the other hand, this disclosure provides a nucleic acid molecule encoding the aforementioned polypeptide.

[0423] The polypeptide, TAFA protein, or its fragments or variants are as described in Section II.

[0424] There are no particular limitations on the nucleic acid molecules applicable to this disclosure, as long as the nucleic acid can be translated into a polypeptide upon transduction into a cell. In some embodiments, the nucleic acid encodes a polypeptide (e.g., a TAFA polypeptide or a polypeptide containing any one of the amino acid sequences of SEQ ID NO: 142 to SEQ ID NO: 147) or a fusion protein.

[0425] In some embodiments, the nucleic acid encodes a protein suitable for preventing or treating a disease or condition (such as those described herein). In some embodiments, the nucleic acid encodes a polypeptide for preventing or treating a specific disease, which is intended to be persistently expressed in a subject or patient.

[0426] In some embodiments, the nucleic acid molecule has at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the nucleic acid encoding the TAFA protein or a fragment or variant thereof.

[0427] In some implementations, the nucleic acid encoding the polypeptide also includes a sequence encoding a signal peptide.

[0428] In some embodiments, the polynucleotides described herein also include regulatory elements. Thus, in some embodiments, the polynucleotides include (1) a regulatory element, (2) a non-translated nucleic acid sequence as described herein (e.g., an EF-1α intron or a fragment thereof), and (3) a transgene (e.g., a nucleic acid molecule encoding a TAFA polypeptide or a polypeptide comprising any one of the amino acid sequences of SEQ ID NO: 142 to SEQ ID NO: 147).

[0429] As used herein, the term "regulatory element" refers to a nucleic acid sequence that regulates (e.g., increases or decreases) the expression of an operatively linked nucleic acid. Regulatory elements suitable for use in this disclosure include, for example, 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, receptor sequences, or combinations thereof. In some embodiments, the regulatory element further comprises sequences for transcription termination (e.g., poly A sequences), sequences for stable transgene expression (e.g., WPRE sequences), sequences for reducing transgene-specific immune development (e.g., miRNA target sequences), or combinations thereof.

[0430] IV. Vectors containing nucleic acids encoding polypeptides

[0431] On the other hand, this disclosure provides a vector containing the aforementioned nucleic acid molecules.

[0432] The nucleic acid molecules are as described in Section III.

[0433] As described herein, such vectors are suitable for recombinant expression in host cells and cells targeted for a therapeutic intervention. In some embodiments, vectors suitable for delivering the polynucleotides described herein (e.g., nucleic acid molecules encoding TAFA peptides or peptides comprising any one of the amino acid sequences of SEQ ID NO: 142 to SEQ ID NO: 147) include viral vectors. Examples of viruses that can be used as vectors in this disclosure include, but are not limited to, retroviruses, herpes simplex virus, lentiviruses, poxviruses, vaccinia virus, rhabdoviruses, adenoviruses, helper-dependent adenoviruses, adeno-associated viruses (AAVs), baculoviruses, and combinations thereof. In some embodiments, vectors that can be used in this disclosure include non-viral vectors. Non-limiting examples of such vectors include plasmids, kinasemids, yeast artificial chromosomes (YACs), bacteriophages, and combinations thereof.

[0434] In some embodiments, the vector comprises one or more sequences selected from promoter sequences, enhancer sequences, exon sequences, intron sequences, signal sequence coding sequences, splice donor sequences, and one or more adeno-associated virus inverted terminal repeat (ITR) sequences.

[0435] On the other hand, this disclosure provides recombinant viral particles comprising the aforementioned vector and capsid protein.

[0436] In a preferred embodiment, the virus may be AAV.

[0437] V. AAV

[0438] In some embodiments, the polynucleotides described herein (e.g., nucleic acid molecules encoding a TAFA polypeptide or a polypeptide containing any one of the amino acid sequences of SEQ ID NO: 142 to SEQ ID NO: 147) are delivered to cells, for example using AAV. Adeno-associated virus (AAV) is a helper-dependent human parvovirus, a single-stranded DNA virus. The AAV genome is approximately 4.7 kbp in size and contains an N-terminal region encoding the rep gene, which is involved in viral replication and viral gene expression; a C-terminal region encoding the cap gene, which encodes the viral capsid protein; and approximately 145 bases of inverted terminal repeats (ITRs) inserted at each end. The 145 bp ITRs have a T-shaped structure and act as the origin of replication during viral genome replication and as the primary packaging signal. The ITR is the only cis-acting sequence required for the preparation of recombinant AAV (rAAV) constructs. The ITR has enhancer activity in the presence of the Rep protein, but its activity is very weak in the absence of the Rep protein. Therefore, when cloning transgenes into recombinant AAV constructs, these characteristics are taken into account, and expression constructs are prepared with appropriate enhancers, promoters, pA, etc. (RJ Samulski and NMuzyczka, Annu. Rev. Virolo. 2014. 1:427-451). Four proteins are translated from the rep gene, named rep78, rep68, rep52, and rep40 according to their molecular weight; they perform important functions in AAV DNA replication. Four proteins are translated from the cap gene; among them, VP1, VP2, and VP3 are structural proteins that constitute AAV particles, and assembly activator protein (AAP) promotes the assembly of structural proteins into AAV particles. For efficient replication of adeno-associated virus, some proteins and RNA from helper viruses such as adenovirus or herpes simplex virus are required (Muzyczka N. Curr Top Microbiol Immunol 158, 97-129, 1992).

[0439] AAV is an attractive vector for delivering transgenes into cells. AAV infection in cultured cells is typically non-cytopathic, and natural infection in humans and other animals is asymptomatic and subclinical. Furthermore, AAV can infect many different types of mammalian cells, thus possessing the potential to target many different tissues in vivo. AAV also has additional advantages that make it a particularly attractive viral system for gene delivery, including inducing a weaker immune response than other forms of gene delivery, and sustained expression from non-integrated episomal vector DNA in both dividing and non-dividing cells. Moreover, AAV is resistant to conditions used for inactivating adenoviruses (e.g., 56°C to 65°C for several hours), making cryogenic storage of rAAV-based vaccines less critical.

[0440] The types of viruses that can be used in this publication include AAVrh.10(AAVrh10), AAV-DJ(AAVDJ), AAV-DJ8(AAVDJ8), AAV1, AAV2, AAV2G9, AAV3, AAV3a, AAV3b, AAV3-3, AAV4, AAV4- 4、AAV5、AAV6、AAV6.1、AAV6.2、AAV6.1.2、AAV7、AAV7.2、AAV8、AAV9、AAV9.11、AAV9.13、AAV9.16、AAV9.24、AAV9.45、AAV9.47、AAV9.61、AAV9.68、AAV 9.84、AAV9.9、AAV10、AAV11、AAV12、AAV16.3、AAV24.1、AAV27.3、AAV42.12、AAV42-1b、AAV42-2、AAV42-3a、AAV42-3b、AAV42-4、AAV42-5a、AAV42-5b、 AAV42-6b、AAV42-8、AAV42-10、AAV42-11、AAV42-12、AAV42-13、AAV42-15、AAV42-aa、AAV43-1、AAV43-12、AAV43-20、AAV43-21、AAV43-23、AAV43-25、AAV43-25 AV43-5、AAV44.1、AAV44.2、AAV44.5、AAV223.1、AAV223.2、AAV223.4、AAV223.5、AAV223.6、AAV223.7、AAV1-7 / rh.48、AAV1-8 / rh.49、AAV2-15 / rh.62 、AAV2-3 / rh.61、AAV2-4 / rh.50、AAV2-5 / rh.51、AAV3.1 / hu.6、AAV3.1 / hu.9、AAV3-9 / rh.52、AAV3-11 / rh.53、AAV4-8 / rh.54、AAV4-9 / rh.54、AAV4-19 / rh.55、AAV5-3 / rh.57、AAV5-22 / rh.58、AAV7.3 / hu.7、AAV16.8 / hu.10、AAV16.12 / hu.11、AAV29.3 / bb.1、AAV29.5 / bb.2、AAV106.1 / hu.37、AAV114.3 / hu.40、AAV127.2 / hu.41、AAV127.5 / hu.42、AAV128.3 / hu.44、AAV130.4 / hu.48、AAV145.1 / hu.53、AAV145.5 / hu.54、AAV145.6 / hu.55、AAV161.10 / hu.60、AAV161.6 / hu.61、AAV33.12 / hu.17、AAV33.4 / hu.15、AAV33.8 / hu.16、AAV52 / hu.19、AAV52.1 / hu.20、AAV58.2 / hu.25、AAVA3.3、AAVA3.4、AAVA3.5、AAVA3.7、AAVC1、AAVC2、AAVC5、AAVF3、AAVF5、AAVH2、AAVrh.72、AAVhu.8、AAVrh.68、AAVrh.70、AAVpi.1、AAVpi.3、AAVpi.2、AAVrh.60、AAVrh.44、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, AAVrh8R A586R variant, AAVrh8R R533A variant, AAAV, BAAV, goat AAV, bovine AAV, AAVhE!1.1, AAVhEr1.5, AAVhER1.14, AAVhEr1.14, AAVhEr1.8, AAVhEr1.16, AAVhEr1.18, AAVhEr1.35, AAVhEr1.7, AAVhEr1.36, AAVhEr2.29, AAVhEr2.4, AAVhEr2.16, AAVhEr2.16, AAVhEr2.30, AAVhEr2.31, AAVhEr2.31, AAVhEr2.36, AAVhER1.23, AAVhEr3.1, AAV2.5T, AAV-PAEC, AAV-LK01, AAV-LK02, AAV-LK03, AAV-LK04, AAV-LK05, AAV-LK06, AAV-LK07, A AV-LK08, AAV-LK09, AAV-LK10, AAV-LK11, AAV-LK12, AAV-LK13, AAV-LK14, AAV-LK15, AAV-L K16, AAV-LK17, AAV-LK18, AAV-LK19, AAV-PAEC2, AAV-PAEC4, AAV-PAEC6, AAV-PAEC7, AAV-PAEC8, AAV-PAEC11, AAV-PAEC12, AAV-2-pre-miRNA-101, AAV-8h, AAV-8b, AAV-h, AAV-b, AAV SM 10-2, AAV Shuffle 100-1, AAV Shuffle 100-3, AAV Shuffle 100-7, AAV Shuffle 10-2, AAV Shuffle 10-6, AAV Shuffle 10-8, AAV Shuffle 100-2, AAV SM 10-1, AAV SM 10-8, AAV SM 100-3, AAV SM 100-10, B P61 AAV, B P62 AAV, B P63 AAV, AAVrh.50, AAVrh.43, AAVrh.62, AAVrh.48, AAVhu.19, AAVhu.11, AAVhu.53, AAV4-8 / rh.64, AAVLG-9 / hu.39, AAV54.5 / hu.2 3. AAV54.2 / hu.22, AAV54.7 / hu.24, AAV54.1 / hu.21, AAV54.4R / hu.27, AAV46.2 / hu.28, AAV46.6 / hu.29, AAV128.1 / hu.43, true type AAV (ttAAV), UPENN AAV10 and Japanese AAV10 serotypes. .

[0441] In some embodiments, the adeno-associated virus (AAV) serotype is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, or AAVrh10. In some embodiments, the AAV serotype is AAV2. In some embodiments, the AAV serotype is AAV5. In some embodiments, the AAV serotype is AAV8. In some embodiments, the AAV serotype is AAV9.

[0442] In some embodiments, for efficient expression or production of adeno-associated virus (AAV), or for other purposes such as retargeting, one or more amino acid sequences of the Rep and / or Cap proteins may be mutated, or new amino acid sequences may be added or deleted, and the sequences of the genes encoding them may also be mutated. Such modified AAVs are included within the scope of the AAVs disclosed herein, provided they retain AAV functionality.

[0443] VI. Cells

[0444] In some embodiments, this disclosure provides cells comprising any of the polynucleotides described herein (e.g., a nucleic acid molecule encoding a TAFA polypeptide or a polypeptide comprising any of the amino acid sequences of SEQ ID NO: 142 to SEQ ID NO: 147) or cells comprising a carrier containing such polynucleotides.

[0445] The nucleic acid molecule is as described in Section III, and the vector is as described in Section IV.

[0446] For example, in some embodiments, cells are transduced, transfected, or transformed using a nucleic acid molecule containing the transgene described herein (e.g., a polypeptide encoding a TAFA peptide or a polypeptide containing any one of the amino acid sequences of SEQ ID NO: 142 to SEQ ID NO: 147) and a vector (e.g., an AAV vector) for expressing the transgene.

[0447] Without intending to be bound by any particular theory, in some embodiments, the cells described herein (e.g., cells transduced with polynucleotides containing a non-translated nucleic acid sequence) are suitable for producing proteins encoded by the transgene described herein (e.g., TAFA peptides or peptides containing any one of the amino acid sequences of SEQ ID NO: 142 to SEQ ID NO: 147). As described herein, in some embodiments, the non-translated nucleic acid sequence (e.g., EF-1α introns or fragments thereof) can enhance the expression of the protein encoded by the transgene in the cell. Therefore, in some embodiments, the cells described herein (e.g., cells transduced with polynucleotides containing a transgene and a non-translated nucleic acid sequence) exhibit higher expression of the encoded protein compared to reference cells. In some embodiments, reference cells are transduced with a corresponding polynucleotide lacking the said non-translated nucleic acid sequence.

[0448] In some embodiments, the cells described herein can produce proteins encoded in vitro. In some embodiments, the cells described herein can produce proteins encoded in vivo (e.g., in a subject receiving the polynucleotides described herein). In some embodiments, the cells described herein can produce proteins encoded both in vitro and in vivo.

[0449] In some embodiments, cells that can be used to produce proteins encoded by transgenes (e.g., in vitro) include host cells. As used herein, the term "host cell" is intended to include cells of any organism that can be transduced with an expression construct or vector (e.g., an AAV vector) to replicate the expression construct or express a gene encoded by the expression construct. Such cells include eukaryotic and prokaryotic cells. As used herein, the term "transduction" is intended to include transfection and transformation. Host cells can be transduced, transfected, or transformed with an expression construct, which refers to the process of delivering or introducing exogenous nucleic acid molecules into a host cell. In some embodiments, the host cell is an isolated host cell containing an AAV vector. In some embodiments, the host cell is an isolated host cell transformed with an AAV vector.

[0450] In some embodiments, the host cell is a eukaryotic cell. In some embodiments, the host cell is selected from mammalian cells, insect cells, yeast cells, transgenic mammalian cells, and plant cells. In some embodiments, the host cell is a prokaryotic cell. In some embodiments, the prokaryotic cell is a bacterial cell.

[0451] In some embodiments, the host cell is an insect cell. In some embodiments, the insect cell is an Sf9 cell. In some embodiments, the host cell is a mammalian cell. Non-limiting examples of mammalian cells that can be used in this disclosure include HEK293, HeLa, ARPE-19, RPE-1, HepG2, Hep3B, Huh-7, C8D1a, Neuro2A, CHO, MES13, BHK-21, COS7, COP5, A549, MCF-7, HC70, HCC1428, BT-549, PC3, LNCaP, Capan-1, Panc-1, MIA PaCa-2, SW480, HCT166, LoVo, A172, MKN-45, MKN-74, Kato-III, NCI-N87, HT-144, SK-MEL-2, SH-SY5Y, C6, HT-22, PC-12, NIH3T3 cells, and combinations thereof.

[0452] In some embodiments, cells that can be used to produce proteins encoded by the transgene (e.g., in vivo) include human cells. In some embodiments, the human cell is the cell of the object to which the nucleic acid molecules described herein are administered. In some embodiments, the human cell is derived from a donor (e.g., a healthy human object).

[0453] In some embodiments, this disclosure provides compositions comprising an AAV vector or host cells comprising an AAV vector or transformed with an AAV vector.

[0454] VII. Composition

[0455] In another aspect, this disclosure provides compositions comprising the polypeptide, a nucleic acid molecule encoding the polypeptide, a vector comprising the nucleic acid molecule, a recombinant viral particle comprising the vector and a capsid protein, a cell comprising the nucleic acid molecule or the vector, or a combination thereof.

[0456] The polypeptides, nucleic acid molecules, vectors, recombinant viral particles, and cells are as described in Sections II through VI.

[0457] In some embodiments, the composition is a pharmaceutical composition.

[0458] In some embodiments, this disclosure provides pharmaceutical compositions comprising (a) a polypeptide described herein (e.g., a TAFA polypeptide or a polypeptide comprising any one of the amino acid sequences of SEQ ID NO: 142 to SEQ ID NO: 147) or a nucleic acid molecule encoding it, and (b) one or more pharmaceutically acceptable carriers. In some embodiments, this disclosure provides pharmaceutical compositions comprising (a) a vector described herein (e.g., rAAV) or recombinant viral particles, and (b) one or more pharmaceutically acceptable carriers. In some embodiments, this disclosure provides pharmaceutical compositions comprising (a) cells described herein, and (b) one or more pharmaceutically acceptable carriers.

[0459] In some embodiments, the pharmaceutical compositions described herein comprise a polypeptide consisting of 8 to 61 amino acid residues.

[0460] In some embodiments, the pharmaceutical composition described herein comprises a polypeptide having an amino acid sequence having at least 50% sequence identity with the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 15 or SEQ ID NO: 87.

[0461] In some embodiments, the pharmaceutical composition is a pharmaceutical composition for the prevention, improvement, or treatment of retinal neurodegenerative diseases.

[0462] In some embodiments, the retinal neurodegenerative disease is a disease caused by total or partial damage to the retina or macula. In some embodiments, the retinal neurodegenerative disease is a disease caused by dysfunction or damage to retinal or macular cells. In some embodiments, the retinal neurodegenerative disease may be a disease caused by dysfunction or damage to photoreceptor cells (e.g., rod and cone cell layers) and / or retinal pigment epithelium (RPE) cells in the retina or macula. In some embodiments, the retinal neurodegenerative disease is retinopathy, choroidal neovascularization, macular degeneration, retinal degeneration, macular edema, retinal swelling, macular swelling, retinal color vision abnormalities, retinal cell degeneration, retinal vascular occlusion, retinal detachment, hereditary retinal diseases, or combinations thereof. In some embodiments, the macular degeneration is age-related macular degeneration, Best macular dystrophy, Sorsby dystrophy, familial dominant drusen, Doyne celluloid retinal dystrophy, Stargardt's disease (Stargardt's macular dystrophy), myopic macular degeneration, or pigment epithelium detachment-related macular degeneration. In some embodiments, the macular degeneration is age-related macular degeneration. In some embodiments, the age-related macular degeneration is wet age-related macular degeneration or dry age-related macular degeneration. In some embodiments, the retinopathy is retinal dystrophy. In some embodiments, the retinopathy is diabetic retinopathy. In some embodiments, the diabetic retinopathy is nonproliferative diabetic retinopathy (NPDR), proliferative diabetic retinopathy (PDR), diabetic macular degeneration, diabetic macular edema, or a combination thereof. In some embodiments, the retinal neurodegenerative disease is a hereditary retinal disease. In some embodiments, the color vision deficiency is color blindness or color weakness. In some embodiments, the color vision abnormality is deuteranopia, protanopia, tritanopia, deuteranomaly, protanomaly, or tritanomaly. In some embodiments, the color vision abnormality is achromatopsia, trichromatic abnormality, or total color blindness. In one embodiment, the polypeptide or nucleic acid molecule encoding it disclosed is suitable for preventing or treating color vision abnormalities by reversing or restoring the destruction, loss, or dysfunction of a photoreceptor, namely a cone cell.

[0463] In some embodiments, the hereditary retinal disease is retinitis pigmentosa (RP), Leber congenital amaurosis, Stargardt's disease, outer exudative retinopathy, cone dystrophy, achoroidal dystrophy, Usher syndrome, Best's disease, X-linked retinoschisis, hereditary color vision abnormalities, or unspecified hereditary retinal dystrophy.

[0464] In some embodiments, the pharmaceutical composition is a pharmaceutical composition for the prevention, improvement, or treatment of neuropathic pain.

[0465] In some embodiments, the neuropathic pain is central nervous system pain, i.e., pain caused by injury or damage affecting all levels of the central nervous system (including the central somatosensory nervous system), such as pain caused by brain injury or spinal cord injury, or pain caused by or associated with diseases or conditions such as epilepsy, multiple sclerosis, or lateral medullary infarction. In some embodiments, central nervous system pain may be spontaneous or stimulus-induced. In some embodiments, central nervous system pain may include mechanical abnormal pain and cold abnormal pain. Symptoms of central nervous system pain typically include, for example, burning, aching, tingling, pressure, painful cold, paresthesia, and sensory disturbances (e.g., tingling, pinprickling, cold, and pressure). The distribution of central nervous system pain may include, for example, a region ranging from a small area to a wide area in the peripheral region, or, in the case of spinal cord injury or stroke, an area covering half of the body, including one side of the face, or the side of the body or limb opposite the lesion. Central nervous system pain resulting from spinal cord injury includes "at-level pain," which is perceived in a segmental pattern at the level of injury, and "below-level pain," which is perceived below the level of injury. In some embodiments, the methods described herein reduce, reverse, alleviate, improve, inhibit, weaken, or prevent central nervous system pain, pain-related symptoms, the underlying cause of pain, or any combination thereof.

[0466] In some embodiments, the neuropathic pain is peripheral neuropathic pain, i.e., pain caused or associated with damage or impairment affecting any part of the peripheral nervous system (e.g., damage to motor nerves, sensory nerves, autonomic nerves, or any combination thereof), or pain caused or associated with a disease or condition. Damage or impairment of motor nerves is associated with symptoms such as muscle weakness (e.g., weakness of muscles in the back, legs, hips, or face), painful spasms, fasciculations (uncontrolled subcutaneous muscle twitching), muscle atrophy (significant reduction in muscle mass), and diminished reflexes. Damage or impairment of sensory nerves causes a variety of symptoms, including pain and hypersensitivity of pain receptors in the skin, leading to abnormal pain (e.g., severe pain induced by normally painless stimuli).

[0467] In some embodiments, the neuropathic pain treatable with the compositions of this disclosure is neuralgia, including but not limited to trigeminal neuralgia (TN) (e.g., pain in the trigeminal nerve region of the face or mouth), atypical trigeminal neuralgia (ATN), occipital neuralgia, postherpetic neuralgia (e.g., unilateral pain distributed in one or more dermatomes or in the ophthalmic branch of the trigeminal nerve), peripheral nerve injury pain (e.g., pain in the area innervated by the damaged nerve, typically distal pain in the area innervated by the damaged nerve in response to trauma, surgery, or compression), glossopharyngeal neuralgia (e.g., severe pain in the throat, tongue, and postauricular region caused by stimulation of the ninth cranial nerve), sciatica, low back pain, and atypical facial pain. In some embodiments, the neuropathic pain is caused by or associated with chemical irritation, inflammation, trauma (including surgery), compression of the nerve by adjacent structures (e.g., tumors), infection, or a combination thereof. In some embodiments, the neuropathic pain is a deafferent neuralgia syndrome, including but not limited to pain caused by brain or spinal cord injury, post-stroke pain, phantom limb pain, paraplegia, brachial plexus avulsion injury, and lumbar radiculopathy. In some embodiments, the neuropathic pain is complex regional pain syndrome (CRPS), including but not limited to CRPS1 and CRPS2. In some embodiments, symptoms associated with CRPS include severe pain, changes in the nails, bones, and skin, and increased sensitivity to touch in the affected limb. In some embodiments, the neuropathic pain is neuropathy (e.g., central or peripheral). Non-limiting examples of neuropathic pain include mononeuropathy and polyneuropathy. In some embodiments, the neuropathic pain is diabetic peripheral neuropathy.

[0468] In some embodiments, the compositions of this disclosure prevent, improve, or treat hyperalgesia. As used herein, the term "hyperalgesia" refers to an increased or exaggerated response to painful stimuli (e.g., needle pricks or hot plates). In some embodiments, hyperalgesia involves mechanical stimuli such as needle pricks (mechanical hyperalgesia). In other embodiments, hyperalgesia involves thermal stimuli such as hot plates (thermal hyperalgesia).

[0469] Pharmaceutically acceptable carriers suitable for use in this disclosure are those commonly used in formulations. Examples of pharmaceutically acceptable carriers include, but are not limited to, lactose, glucose, sucrose, sorbitol, mannitol, starch, gum arabic, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methylcellulose, methylparaben, propylparaben, talc, magnesium stearate, and mineral oil. The pharmaceutical compositions of this disclosure may also contain one or more additives selected from lubricants, wetting agents, sweeteners, flavoring agents, emulsifiers, suspending agents, and preservatives. Details of suitable pharmaceutically acceptable carriers and formulations can be found in Remington's Pharmaceutical Sciences (19th edition, 1995).

[0470] The pharmaceutical compositions disclosed herein are formulated to be compatible with their intended route of administration. Suitable non-oral routes of administration include, for example, intravenous injection, transdermal administration, subcutaneous injection, intramuscular injection, intraocular (e.g., subcapsular, subconjunctival, suprachoroidal, subchoroidal space, subretinal, vitreous, and any other intraocular route of administration capable of delivering the composition to a similar location), local ocular, intravenous, intrasheath, intraamniotic, intraarticular, intraarticular, intracardiac, intracavitary, intracerebral, intracisional, intracoronary, intracranial, intradural, epidural, intrahippocampal, intranasal, intraosseous, intraperitoneal, intrapleural, intraspinal, intrathoracic, intrathymic, intrauterine, intravaginal, intraventricular, intrabladder, subconjunctival, intratumoral, local, intraperitoneal, and combinations thereof. In some aspects, intraocular administration includes suprachoroidal, subretinal, and intravitreal administration. In some aspects, local administration includes local ocular, intranasal, transdermal, oral, or rectal administration. Since the amino acid sequence of the disclosed polypeptide (e.g., TAFA protein fragment or variant thereof) can consist of 8 to 61 amino acids, preferably 8 to 30 amino acids, the intended preventive or therapeutic effects can be achieved by topical ocular or intranasal administration.

[0471] In some embodiments, the pharmaceutical composition is administered at a daily dose of 0.0001 mg / kg to 100 mg / kg.

[0472] The pharmaceutical compositions disclosed herein can be formulated with one or more pharmaceutically acceptable carriers and / or excipients. The compositions can be provided in unit dose form or dispensed in multi-dose containers. Formulations can be in the form of solutions, suspensions, or emulsions in oily or aqueous media, or in the form of extracts, powders, granules, tablets, or capsules. Formulations may also contain dispersants or stabilizers.

[0473] VIII. Reagent Kit

[0474] This disclosure also provides kits comprising one or more polypeptides as described herein (e.g., a TAFA polypeptide or a polypeptide comprising any one of the amino acid sequences of SEQ ID NO: 142 to SEQ ID NO: 147), one or more polynucleotides (e.g., comprising a transgenic and untranslated nucleic acid sequence), one or more vectors as described herein (e.g., an AAV vector), one or more cells as described herein (e.g., comprising an AAV vector or a host cell transformed with an AAV vector), any composition described herein, or any combination thereof. In some embodiments, the kits also include instructions for use.

[0475] As used herein, the terms “reagent kit” and “system” in some embodiments are intended to refer to at least one or more polynucleotides described herein, one or more vectors described herein (e.g., AAV vectors), one or more host cells described herein, any pharmaceutical composition described herein, or any combination thereof, optionally in combination with one or more additional types of elements or components (e.g., other biochemical reagents, containers, packaging such as packaging intended for commercial sale, instructions for use, etc.).

[0476] IX. Uses and Methods

[0477] IX.A. Production Method

[0478] This disclosure also provides methods for producing a combination of the polypeptide (e.g., a TAFA polypeptide or a polypeptide containing any one of the amino acid sequences of SEQ ID NO: 142 to SEQ ID NO: 147), a nucleic acid molecule encoding the polypeptide, a vector containing the nucleic acid molecule, a recombinant viral particle containing the vector and a capsid protein, a cell containing the vector, a cell transformed with the vector, or any combination thereof.

[0479] In some implementations, such methods include synthesizing the TAFA protein fragment or a variant thereof described herein under suitable conditions.

[0480] In some embodiments, such methods include culturing the cells described herein (e.g., cells transduced with a polynucleotide containing a transgenic and untranslated nucleic acid molecule) under suitable conditions and recovering the encoded protein. In some embodiments, methods for producing polypeptides encoded by a transgenic gene include administering a polynucleotide of this disclosure (e.g., containing a transgenic and untranslated nucleic acid molecule) to a subject of need, such that the encoded polypeptide is produced in the subject. Further disclosures regarding such methods of producing polypeptides in vivo are provided elsewhere in this disclosure (see, for example, therapeutic uses below).

[0481] In some embodiments, this disclosure provides methods for producing recombinant adeno-associated virus (rAAV) particles comprising the polynucleotides described herein (e.g., transgenic and untranslated nucleic acid sequences). In some embodiments, the method for producing such recombinant AAV includes culturing cells transfected with the AAV vector described herein under conditions suitable for producing recombinant AAV. In some embodiments, the method further includes the step of isolating the produced recombinant viral particles.

[0482] In some embodiments, this disclosure provides recombinant viral particles produced by the method described herein.

[0483] In some embodiments, recombinant viral particles can be produced by transfecting cells with (i) an AAV vector containing a transgene (e.g., a nucleic acid molecule encoding a polypeptide or a polypeptide containing any one of the amino acid sequences of SEQ ID NO: 142 to SEQ ID NO: 147) and (ii) a construct containing the rep and cap genes. Additionally, (iii) helper constructs for transducing the transgene into host cells can be used. In such embodiments, the helper construct may contain an E2A gene that promotes AAV genome replication and AAV gene transcription, an E4 gene that allows AAV mRNA to move from the nucleus to the cytoplasm, and a VA region that generates two VA RNAs involved in translation control.

[0484] In some implementations, the three constructs described above can be replaced by two constructs for transducing host cells. In such implementations, the AAV construct contains a transgenic and untranslated nucleic acid sequence, while the individual constructs contain rep and cap genes, E2A gene, E4 gene, and VA region. Other methods for producing AAV particles are well known in the art and described, for example, in Clement et al., Mol Ther Methods Clin Dev 3:16002 (2016), Clark, Kidney Int. 61:S9-15 (2002), and Xiao et al., J Virol 72(3):2224-32 (1998), each of which is incorporated herein by reference in its entirety.

[0485] This disclosure also provides recombinant viral particles comprising (a) a capsid protein and (b) an AAV vector.

[0486] IX.B. Therapeutic Use

[0487] The polypeptides described herein (e.g., TAFA polypeptides or polypeptides comprising any one of the amino acid sequences of SEQ ID NO: 142 to SEQ ID NO: 147), nucleic acids (e.g., comprising transgenic and untranslated nucleic acid sequences), vectors carrying such nucleic acids, and recombinant viruses (e.g., rAAV), cells comprising said nucleic acids or vectors, and the methods described herein have numerous in vitro and in vivo uses. For example, the polypeptides, polynucleotides, and vectors (e.g., AAV vectors) described herein can be administered to cultured cells (in vitro or ex vivo) or human subjects (in vivo) to prevent or treat diseases. Thus, in some embodiments, this disclosure provides therapeutic uses for any polypeptide, polynucleotide (e.g., comprising transgenic and untranslated nucleic acid sequences), recombinant expression constructs or vectors, cells, pharmaceutical compositions, or recombinant viruses described herein. In some embodiments, this disclosure provides methods for expressing transgenes in a desired subject, including administering to the subject a polynucleotide as disclosed herein (e.g., comprising transgenic and untranslated nucleic acid sequences), a vector as disclosed herein, a recombinant virus as disclosed herein (e.g., rAAV), a cell as disclosed herein, or a pharmaceutical composition as disclosed herein, wherein the expression of the transgene in the subject increases after administration.

[0488] As described herein, when a transgene is translated, the untranslated nucleic acid sequences of this disclosure can increase transgene expression. Therefore, in some embodiments, this disclosure provides a method for increasing transgene expression in cells, comprising contacting cells with any of the polynucleotides described herein (e.g., nucleic acid molecules encoding a TAFA polypeptide or a polypeptide comprising any one of the amino acid sequences of SEQ ID NO: 142 to SEQ ID NO: 147), a vector, or a recombinant virus (e.g., rAAV). Contact can be performed in vitro or in vivo. When contact is performed in vivo, the method may further include administering any of the polynucleotides, vectors, or recombinant viruses described herein to the subject prior to contact.

[0489] In some embodiments, following such exposure, the expression of the transgene (e.g., a nucleic acid molecule encoding a TAFA peptide or a peptide comprising any one of the amino acid sequences of SEQ ID NO: 142 to SEQ ID NO: 147) increases by at least about 1-fold, at least about 1.1-fold, at least about 1.2-fold, at least about 1.3-fold, at least about 1.4-fold, at least about 1.5-fold, at least about 1.6-fold, at least about 1.7-fold, at least about 1.8-fold, at least about 1.9-fold, at least about 2-fold, at least about 2.5-fold, at least about 3-fold, at least about 3.5-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, or at least about 10-fold compared to baseline levels. In some embodiments, baseline expression is the expression of the transgene in cells prior to exposure. In some embodiments, baseline expression is the expression of the transgene in the corresponding cells that have not yet been exposed to the peptide, polynucleotide, vector, or recombinant virus described herein.

[0490] Another aspect of this disclosure provides a method for preventing or treating a disease in a subject of need, comprising administering to the subject an effective amount of any polypeptide described herein (e.g., a TAFA polypeptide or a polypeptide comprising any one of the amino acid sequences of SEQ ID NO: 142 to SEQ ID NO: 147), a polynucleotide (e.g., a nucleic acid molecule encoding a TAFA polypeptide or a polypeptide comprising any one of the amino acid sequences of SEQ ID NO: 142 to SEQ ID NO: 147), a vector, a cell, a recombinant virus, or a pharmaceutical composition. It will be apparent from this disclosure that the compositions described herein (e.g., polypeptides, polynucleotides, recombinant expression constructs, cells, pharmaceutical compositions, or recombinant viruses) can be used to prevent or treat any disease of interest.

[0491] In some embodiments, the method further includes administering an additional therapeutic agent (e.g., a vascular endothelial growth factor (VEGF) inhibitor or a therapeutic agent for neuropathic pain) to the subject. In some embodiments, the additional therapeutic agent may be administered before, simultaneously with, or after the administration of a peptide, polynucleotide, vector, cell, recombinant virus, or pharmaceutical composition.

[0492] There are no limitations on the diseases that can be prevented, improved, or treated by this disclosure, and it includes any disease for which there is a desire to reduce the frequency of application. Non-limiting examples of such diseases include retinal neurodegenerative diseases. In some aspects, said retinal neurodegenerative diseases are selected from retinopathy, choroidal neovascularization, macular degeneration, retinal degeneration, macular edema, retinal swelling, macular swelling, retinal edema, color vision abnormalities, retinal cell degeneration, retinal vascular occlusion, retinal detachment, hereditary retinal diseases, and combinations thereof.

[0493] In some aspects, retinal neurodegenerative diseases that can be prevented or treated by this disclosure include macular degeneration. In some aspects, said macular degeneration includes age-related macular degeneration (AMD). Age-related macular degeneration can be classified as dry (atrophic) macular degeneration and wet (neovascular or exudative) macular degeneration. Age-related macular degeneration can also be classified as early AMD, intermediate AMD, and late or terminal AMD (geographic atrophy). In some aspects, retinal or macular diseases that can be prevented or treated by this disclosure include diabetic retinopathy. In some aspects, said diabetic retinopathy is non-proliferative diabetic retinopathy (NPDR). In some aspects, said diabetic retinopathy is proliferative diabetic retinopathy (PDR). In some aspects, said diabetic retinopathy is diabetic macular degeneration. In some aspects, said diabetic retinopathy is diabetic macular edema. In some aspects, said diabetic retinopathy is any retinal disease associated with ischemic damage within the retina. Unless otherwise stated, this disclosure can be used for the prevention or treatment of all forms of AMD and / or diabetic retinopathy.

[0494] Additional, non-limiting examples of the disease include neuropathic pain.

[0495] In some embodiments, the neuropathic pain is central nervous system pain, i.e., pain caused by injury or damage affecting all levels of the central nervous system (including the central somatosensory nervous system), such as brain injury or spinal cord injury, or pain caused by or associated with diseases or conditions such as epilepsy, multiple sclerosis, or lateral medullary infarction. In some embodiments, central nervous system pain may be spontaneous or stimulus-induced. In some embodiments, central nervous system pain may include mechanical abnormal pain and cold abnormal pain. Symptoms of central nervous system pain typically include, for example, burning, aching, tingling, pressure, painful cold, paresthesia, and sensory disturbances (e.g., tingling, pinprickling, cold, and pressure). The distribution of central nervous system pain may include, for example, an area ranging from a small area in the peripheral region to a wide area, or, in the case of spinal cord injury or stroke, an area covering half of the body, including one side of the face or the side of the body or limb opposite the lesion. Central nervous system pain caused by spinal cord injury includes "horizontal pain," which is perceived in a segmental pattern at the level of injury, and "sub-horizontal pain," which is perceived below the level of injury. In some embodiments, the methods described herein reduce, reverse, alleviate, improve, inhibit, weaken, or prevent central nervous system pain, pain-related symptoms, the underlying cause of pain, or any combination thereof.

[0496] In some embodiments, the neuropathic pain is peripheral neuropathic pain, i.e., pain caused or associated with damage or impairment affecting any part of the peripheral nervous system (e.g., damage to motor nerves, sensory nerves, autonomic nerves, or any combination thereof), or pain caused or associated with a disease or condition. Damage or impairment of motor nerves is associated with symptoms such as muscle weakness (e.g., weakness of muscles in the back, legs, hips, or face), painful spasms, fasciculations (uncontrolled subcutaneous muscle twitching), muscle atrophy (significant reduction in muscle mass), and diminished reflexes. Damage or impairment of sensory nerves causes a variety of symptoms, including pain and hypersensitivity of pain receptors in the skin, leading to abnormal pain (e.g., severe pain induced by normally painless stimuli).

[0497] In some embodiments, the method of the present invention treats one or more types of neuropathic pain by administering the polypeptide, a nucleic acid molecule encoding the polypeptide, a carrier containing the nucleic acid molecule, a recombinant viral particle containing the carrier and a capsid protein, a cell containing the carrier, a cell transformed with the carrier, or any combination thereof to a subject in need. In some embodiments, the neuropathic pain treatable by the method of the present invention is neuralgia, including but not limited to trigeminal neuralgia (TN) (e.g., pain in the trigeminal nerve region of the face or mouth), atypical trigeminal neuralgia (ATN), occipital neuralgia, postherpetic neuralgia (e.g., unilateral pain distributed in one or more dermatomes or in the ophthalmic branch of the trigeminal nerve), peripheral nerve injury pain (e.g., pain in the area innervated by the damaged nerve, typically distal pain in the area innervated by the damaged nerve in response to trauma, surgery, or compression), glossopharyngeal neuralgia (e.g., severe pain in the throat, tongue, and postauricular region caused by stimulation of the ninth cranial nerve), sciatica, low back pain, and atypical facial pain. In some embodiments, neuropathic pain is caused by or associated with chemical irritation, inflammation, trauma (including surgery), compression of the nerve by adjacent structures (e.g., tumors), infection, or a combination thereof. In some embodiments, the neuropathic pain is a deafferent neuralgia syndrome, including but not limited to brain or spinal cord injury, post-stroke pain, phantom limb pain, paraplegia, brachial plexus avulsion injury, and lumbar radiculopathy. In some embodiments, the neuropathic pain is a complex regional pain syndrome (CRPS), including but not limited to CRPS1 and CRPS2. In some embodiments, symptoms associated with CRPS include severe pain, changes in the nails, bones, and skin, and increased sensitivity to touch in the affected limb. In some embodiments, the neuropathic pain is a neuropathy (e.g., central or peripheral). Non-limiting examples of neuropathic pain include mononeuropathy (mononeuropathy) and polyneuropathy (polyneuropathy).

[0498] In some embodiments, the neuropathic pain is caused by or associated with physical injury, including, for example: (1) traumatic injury or damage, including nerve compression (e.g., nerve squeezing, nerve stretching, nerve entrapment, or partial nerve transection); (2) spinal cord injury (e.g., hemisection); (3) injury or damage to peripheral nerves (e.g., motor nerves, sensory nerves, or autonomic nerves, or any combination thereof); (4) limb amputation; contusion; inflammation (e.g., myelitis); or surgery; and (5) repetitive stress, such as repetitive, slow, and / or forceful activities requiring prolonged joint movement (e.g., ulnar neuropathy and carpal tunnel syndrome). In some embodiments, the method treats neuropathic pain caused by or associated with exposure to a toxic agent.

[0499] In some embodiments, the neuropathic pain is caused by or associated with one or more diseases or conditions, including, for example: (1) ischemic events (e.g., stroke or myocardial infarction); (2) multiple sclerosis; (3) metabolic and / or endocrine diseases or conditions (e.g., diabetes, metabolic diseases, and acromegaly caused by excessive production of growth hormone, characterized by abnormal enlargement of skeletal portions including joints, leading to nerve entrapment and pain); (4) small vessel diseases that reduce oxygen supply to peripheral nerves and cause damage to nerve tissue (e.g., vasculitis, i.e., vascular inflammation); and (5) autoimmune diseases (e.g., Sjögren's syndrome, lupus, rheumatoid arthritis, and acute inflammatory demyelination). (6) Polyneuropathy (also known as Guillain-Barré syndrome); (7) Kidney impairment; (8) Cancer or tumor (e.g., neoplastic tumors, neuromas, paraneoplastic syndromes, and toxicity from cancer chemotherapy and radiotherapy); (9) Infections (e.g., varicella-zoster virus, Epstein-Barr virus, West Nile virus, cytomegalovirus, and herpes simplex virus, viral infections such as HIV / AIDS, or bacterial infections such as bacteria that cause Lyme disease and diphtheria, and Mycobacterium leprae that causes leprosy); (10) Inflammatory conditions; (11) Peripheral neuropathy (e.g., neuroma); (12) Hereditary or de novo hereditary conditions (e.g., peroneal muscular atrophy); (13) Mononeuropathy; (14) Polyneuropathy; or combinations thereof. In some embodiments, the neuropathic pain is caused by or associated with diabetes (type I or type II). In some embodiments, the neuropathic pain is diabetic peripheral neuropathy.

[0500] In some embodiments, the neuropathic pain is caused by or associated with exposure to infectious agents such as tick-borne infections, varicella-zoster virus, EB virus, West Nile virus, cytomegalovirus and herpes simplex virus, HIV / AIDS, or exposure to toxic substances such as drugs, alcohol, heavy metals (e.g., lead, arsenic, mercury), industrial substances (e.g., fumes from solvents or adhesives), and nitrous oxide.

[0501] In some embodiments, the neuropathic pain is caused by or associated with physical trauma, infection, diabetes, cancer treatment, alcoholism, amputation, multiple sclerosis, herpes zoster, spinal surgery, sciatica (pain along the sciatic nerve), low back pain, neuralgia such as trigeminal neuralgia (e.g., pain in the trigeminal nerve region of the face or mouth), neuropathic pain such as painful polyneuropathy (e.g., foot pain extending to the calf, thigh, and hand), or a combination thereof. In some embodiments, the neuropathic pain is trigeminal neuralgia. In some embodiments, the neuropathic pain is associated with weakness of the muscles in the back, legs, hips, or face. In some embodiments, the neuropathic pain is caused by nerve compression, for example, nerves in the legs, feet, or hips, or nerves in the facial muscles. In some embodiments, the neuropathic pain involves sciatic nerve injury. In some embodiments, the neuropathic pain is sciatica.

[0502] In some embodiments, the method of the present invention can reverse, alleviate, improve, suppress, reduce, or prevent one or more symptoms associated with neuropathic pain. Therefore, in one aspect, the present invention provides a method for improving hyperalgesia, comprising administering to a subject in need the polypeptide, a nucleic acid molecule encoding the polypeptide, a carrier comprising the nucleic acid molecule, a recombinant viral particle comprising the carrier and a capsid protein, a cell comprising the carrier, a cell transformed with the carrier, or any combination thereof. As used herein, the term “hyperalgesia” refers to an increased or exaggerated response to a painful stimulus (e.g., a needle prick or a hot plate). In some embodiments, hyperalgesia involves mechanical stimulation such as a needle prick (mechanical hyperalgesia). In other embodiments, hyperalgesia involves thermal stimulation such as a hot plate (thermal hyperalgesia). In some embodiments, the subject in need has a chronic compressive injury (e.g., sciatica). In some embodiments, the subject in need has diabetic peripheral neuropathy.

[0503] In some embodiments, when the polypeptide (e.g., TAFA protein; a polypeptide comprising an amino acid sequence of a TAFA protein fragment or a variant thereof), a nucleic acid molecule encoding the polypeptide, a vector comprising the nucleic acid molecule, a recombinant viral particle comprising the vector and a capsid protein, a cell comprising the vector, a cell transformed with the vector, or any combination thereof, is applied to a subject in need (e.g., an untreated subject suffering from neuropathic pain), the subject may exhibit a higher mechanostimulation threshold compared to a reference control. As used herein, the term "mechanostimulation threshold" refers to the amount of pressure from a mechanical stimulus (e.g., tension) required for the subject to respond. Thus, a subject with a higher threshold can withstand or tolerate significantly greater amounts of mechanical stimulation compared to a subject with a lower threshold. In some embodiments, the method of the present invention increases the subject's mechanostimulation threshold by at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 150%, or at least 200% compared to a reference control (e.g., the subject's threshold before application).

[0504] In some embodiments, when the polypeptide, the nucleic acid molecule encoding the polypeptide, the vector containing the nucleic acid molecule, the recombinant viral particle containing the vector and the capsid protein, the cell containing the vector, the cell transformed with the vector, or any combination thereof, is applied to a subject in need, the latency period (i.e., the time interval between stimulation and response) of the thermal stimulus (e.g., a hot plate) is increased compared to a reference control (a subject suffering from neuropathic pain who has not received the treatment). In some embodiments, the method of the present invention increases the latency period of the subject to thermal stimulation by at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 150%, or at least 200% compared to a reference control (e.g., the latency period of the subject before application).

[0505] On the other hand, the present invention provides a method for improving sensory nerve conduction velocity in subjects with a need. As used herein, the term “sensory nerve conduction velocity” (SNCV) refers to the speed at which an electrical signal travels through a peripheral nerve. Healthy nerves transmit electrical signals faster and more strongly than damaged nerves (see, for example, Chouhan S., J Clin Diagn Res 10(1):CC01-3 (2016)). Therefore, tests measuring SNCV (e.g., sensory nerve conduction velocity tests) are suitable for identifying potential nerve damage and / or functional impairment in subjects. In some embodiments, the method of the present invention improves the SNCV of subjects suffering from neuropathic pain by at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 150%, or at least 200% compared to a reference control (e.g., the SNCV of the subject before application).

[0506] Animal models for studying neuropathic pain are available. Non-limiting examples of such animal models include: (1) a spinal nerve ligation (SNL) model in which one or more spinal nerves leading to the paw are ligated and severed (see Kim SH and Chung JM., Pain 50:355-363 (1992)); (2) a partial sciatic nerve ligation (PSL) model in which a portion of the sciatic nerve is tightly ligated (see Seltzer et al., Pain 43:205-218 (1990)); (3) a chronic compression injury (CCI) model in which four loose ligatures made of chromic gut are placed around the sciatic nerve, and an immune response to the sutures induces nerve swelling and contraction; (4) a selective nerve injury (SNI) model in which the common peroneal nerve and tibial nerve are severed while the sural nerve is preserved intact (see Devor I. and Woolf C.J., Pain 87:149-158). (2000)); and (5) STZ-induced diabetic rats, in which injection of streptozotocin (STZ) induces pancreatic edema and degeneration of β-cells in the islets, thereby inducing experimental diabetes in rats (see, for example, Akbarzadeh A. et al., Indian J. Clin. Biochem. 22(2):60-64 (2007)). These models induce hyperalgesia in animals, manifested as enhanced responses to mechanical and / or thermal stimuli.

[0507] Mechanical hyperalgesia in animals can be assessed using the Von Frey test, in which a series of Von Frey monofilaments with different bending forces are applied to the plantar surface of the paw. Following nerve injury, the claw withdrawal threshold decreases sharply (see Li et al., Pain 85:493-502 (2000)). Therefore, in some embodiments, the method of the present invention increases the claw withdrawal threshold in animal models of neuropathic pain (e.g., chronic compression injury models).

[0508] Testing for thermal hyperalgesia may include using a radiant heat source (e.g., a hot plate) focused on the surface of the foot and measuring the claw withdrawal reaction time. Claw withdrawal occurs more rapidly after nerve injury than before injury (see Kim SH and Chung JM., Pain 50:355-363 (1992)). In some embodiments, the method of the present invention increases the claw withdrawal latency in animal models of neuropathic pain (e.g., diabetic peripheral neuropathy models).

[0509] Another aspect of this disclosure provides gene therapy or methods for the prevention or treatment of diseases that enable sustained expression of transgenes.

[0510] Using the viral delivery system described herein, the compositions of this disclosure can be administered 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 more. In some embodiments, the interval is about 2 to about 3 months. In some embodiments, the interval is about 6 months. In some embodiments, the interval is about 1 year. In some embodiments, the interval is at least about 1 year. In some embodiments, the interval is at least about 2 years. In some embodiments, the interval is at least about 3 years. In some embodiments, the interval is at least about 4 years. In some embodiments, the interval is at least about 5 years. In some embodiments, the interval is at least about 10 years. That is, by using the viral delivery system described herein, the frequency of composition administration can be significantly reduced, thereby sparing physicians, patients, or subjects the inconvenience associated with repeated administration. Depending on the patient’s symptoms or needs, the composition may initially be applied 2 to 3 times at intervals of 1 to 2 weeks, then once every 2 to 3 months, every 6 months, or once a year or longer, or once every 2 to 10 years or longer.

[0511] Invention Effects

[0512] The features and advantages of this disclosure can be summarized as follows:

[0513] (i) The present invention provides polypeptides (e.g., polypeptides comprising the amino acid sequence of TAFA protein, TAFA protein fragments or variants thereof) that have the ability to increase neurite length and / or the number of branch points.

[0514] (ii) The present invention also provides therapeutic use of pharmaceutical compositions comprising the polypeptide, nucleic acid molecules encoding the polypeptide, vectors comprising the nucleic acid molecules, recombinant viral particles comprising the vector and capsid protein, cells comprising the vector, cells transformed with the vector, or any combination thereof.

[0515] (iii) By repairing damaged nerves, the compositions disclosed herein can be effectively used to prevent or treat retinal neurodegenerative diseases and neuropathic pain. Attached Figure Description

[0516] Figure 1A This study presents an analysis of changes in neurite length induced by full-length TAFA4 protein in differentiated neurons derived from the mouse dorsal root ganglion progenitor cell line MED17.11. Figure 1B The analysis shows the changes in the number of branch points induced by the full-length TAFA4 protein in the same neuronal cells. An asterisk (*) indicates a p-value < 0.05.

[0517] Figure 2 This study presents an analysis by fluorescence angiography (FA) of the efficacy of subretinal administration of control AAV8 (control group) or AAV8.TAFA4 in mice (test group) in improving retinal damage in a NaIO3-induced retinal injury model. OD (oculus dexter, right eye) represents the right eye, and OS (oculus sinister, left eye) represents the left eye.

[0518] Figure 3 The measurements of A-wave and B-wave amplitude changes in electroretinograms (ERG) are shown between the control group (control AAV8) and the test group (AAV8. mouse TAFA4).

[0519] Figure 4 A comparison of interspecies sequence identity of the full-length TAFA4 protein is shown.

[0520] Figure 5 A comparison of interspecies sequence identity of mature TAFA4 protein is shown.

[0521] Figure 6This study presents an analysis of the efficacy of subretinal administration of control AAV8 (control group) or test group (AAV8.human TAFA4, AAV8.g ...

[0522] Figure 7 The measurements of A-wave and B-wave amplitude changes in electroretinograms (ERG) are shown between the control group (control AAV8) and the test group (AAV8.human TAFA4, AAV8.g.g.g., and AAV8.g.g., fish TAFA4).

[0523] Figure 8 This study presents an analysis of the efficacy of subretinal administration of control AAV8 (control group) or test group (AAV8.TAFA1, AAV8.TAFA2, AAV8.TAFA3, or AAV8.TAFA4) in improving retinal damage in a NaIO3-induced retinal injury model, performed by fluorescence angiography (FA). OD (right eye) represents the right eye, and OS (left eye) represents the left eye.

[0524] Figure 9 The measurements of A-wave and B-wave amplitude changes in electroretinograms (ERG) are shown between the control group (control AAV8) and the test groups (AAV8.TAFA1, AAV8.TAFA2, AAV8.TAFA3 and AAV8.TAFA4).

[0525] Figures 10A to 10D A comparison of the interspecific amino acid sequence identity of full-length TAFA1, TAFA2, TAFA3, and TAFA4 is shown. Figure 10A The amino acid residues 1 to 70 were compared. Figure 10B The amino acid residues 71 to 140 were compared. Figure 10C Amino acid residues 141 to 210 were compared, and Figure 10D Amino acid residues 211 to 244 were compared.

[0526] Figure 11A The analysis shows the changes in neurite length induced by TAFA4 peptide fragments (TAFA4 fragments 1 to 3, F1 to F3) in differentiated neurons derived from the MED17.11 cell line, and Figure 11B The analysis shows the changes in the number of branch points induced by TAFA4 peptide fragments (F1 to F3) in the same cells.

[0527] Figure 12 The sequence of a TAFA4-derived peptide (TAFA protein and its fragments F1 to F8) according to an embodiment of the present invention is shown.

[0528] Figure 13A The analysis shows the changes in neurite length induced by TAFA4 peptide fragments (TAFA4 fragments 4 to 8, F4 to F8) in differentiated neurons derived from the MED17.11 cell line, and Figure 13B The analysis shows the changes in the number of branch points induced by the TAFA4 peptide fragments (F4 to F8) in the same cells. Double asterisks (**) indicate p-values ​​< 0.01.

[0529] Figure 14 An analysis of the analgesic effects of TAFA4 and TAFA4 fragments 1, 2, 3, 5, 6 and 7 (F1, F2, F3, F5, F6 and F7) in a neuropathic pain model is presented. Detailed Implementation

[0530] The present invention will be further described in detail below with reference to embodiments. These embodiments are provided only to illustrate the invention more specifically, and it will be apparent to those skilled in the art that the scope of the invention is not limited to these embodiments in the spirit of the invention.

[0531] Example

[0532] [Example 1] Evaluation of the efficacy of TAFA4 on neuronal cells

[0533] To evaluate the biological efficacy of TAFA4 on neurons, mouse dorsal root ganglion progenitor cell line MED17.11 was differentiated into neurons, and the changes in neurite length and branch point number induced by recombinant human TAFA4 (hereinafter referred to as "rhTAFA4") were evaluated.

[0534] MED17.11 cell lines were cultured in a 33°C incubator (5% CO2). Undifferentiated cells were maintained in DMEM / F 12 medium (Gibco) supplemented with 10% fetal bovine serum (Gibco), 1% penicillin / streptomycin (Gibco), 5 ng / mL interferon-γ (R&D Systems), and 0.5% chicken embryo extract (Sera Lab). The efficacy of TAFA4 was assessed as follows: Undifferentiated MED17.11 cells were harvested using 0.25% trypsin-EDTA (Gibco). After centrifugation, the supernatant was removed, and the cell pellet was resuspended in differentiation-inducing medium. The differentiation-inducing medium consisted of DMEM / F-12 supplemented with 10% fetal bovine serum (Gibco), 1% penicillin / streptomycin (Gibco), 10 ng / mL fibroblast growth factor 2 (R&D Systems), 0.5 mM butyryl cAMP (Sigma), 25 μM forskolin (Sigma), 5 μg / mL Y-27632 (Chemdea), 100 ng / mL β-nerve growth factor (R&D Systems), and 10 ng / mL glial cell-derived neurotrophic factor (R&D Systems). MED17.11 cells resuspended in the differentiation-inducing medium were cultured at 1 × 10⁻⁶ cells / mL. 4 Cells / mL, 100 μL per well, were randomly assigned to 96-well cell culture plates (Thermo Fisher). The test group was treated with 1 μM rhTAFA4 (R&D Systems, Table 1), while the control group (CTL) was treated with an equal volume of phosphate-buffered saline (PBS) (Gibco). Both groups were then incubated at 37°C (5% CO2). Cell images were acquired using the Incucyte system (Sartorius) during incubation. The neurite length and branching point number in MED17.11 cells were analyzed from the acquired images using Incucyte software.

[0535] Three independent experiments were conducted in total, and statistical significance was analyzed using Student's t-test.

[0536] [Table 1]

[0537] Amino acid sequence of recombinant human TAFA4 peptide

[0538]

[0539] Four days after treatment, compared with the control group (CTL), the rhTAFA4 treatment group showed a significant increase in neurite length and branching point number of MED17.11 cells. Figure 1A and Figure 1B ).

[0540] [Example 2] Efficacy evaluation of TAFA in a retinal injury model

[0541] 2-A. Production of AAVs carrying TAFA1 to TAFA4 and interspecific variant genes

[0542] The method described in Korean Patent Application No. 10-2023-0068976 produces a recombinant AAV containing a transgene, and inserts genes encoding TAFA1 to TAFA4 and their interspecific variants as transgenes.

[0543] 2-B. Efficacy evaluation of TAFA4 in a NaIO3 (sodium iodate)-induced retinal injury model

[0544] It has been reported that NaIO3 (sodium iodate) induces direct damage to retinal neurons and reduces the area of ​​neurites and retinal ganglion cells (see Zui Tao et al., Molecular Neurobiology, 2013 Feb; 47 (1): 241-60).

[0545] As confirmed in Example 1 above, TAFA4 significantly increased neurite length and the number of branch points compared to the control. To assess whether TAFA4 improves retinal neurodegenerative diseases in an in vivo model, changes in fluorescence angiography (FA) and electroretinography (ERG) after administration of AAV8 to TAFA4 were evaluated in a NaIO3-induced retinal injury mouse model.

[0546] C57BL / 6 mice (Orient Bio) were administered non-transgenic control AAV8 or AAV8 mouse TAFA4 (CAT311 promoter-mTAFA4) via subretinal injection (SRI).

[0547] To allow for adequate TAFA4 expression in mice, 5 × 10⁵ t / v was administered to both eyes of each mouse via subretinal injection (SRI). 8Mice were injected with AAV8 via vg and maintained for 56 days. An AMD model was then induced by tail vein injection of NaIO3 at a dose of 20 mg / kg. Ten days after model induction (day 66 after SRI), a fluorescent contrast agent was injected via tail vein. Images were focused on the fundus using a Micron-IV imaging camera (Phoenix), and fluorescence angiography (FA) images were acquired. Eleven days after model induction (day 67 after SRI), dark-adapted ERG was performed. Mice were placed on an ERG stage, and ERG probes were applied to the tail, head, and cornea to measure A-wave and B-wave amplitudes. ERG data were analyzed using the LabScribeERG (iWorx Data Acquisition Software) program.

[0548] In FA images acquired 10 days after model induction, contrast agent leakage due to outer retinal damage was observed in the retinas of mice treated with NaIO3 (control AAV8 treatment) compared to the uninduced AMD healthy group (initial group, mice treated with control AAV8). Conversely, the group treated with AAV8 and mouse TAFA4 (test group) showed almost normal retinal manifestations similar to the initial group. Figure 2 Consistent with the findings of FA, dark-adapted ERG assessed on day 11 after model induction showed NaIO3-induced retinal abnormalities, with significantly reduced amplitudes of induced A and B waves. Conversely, in the group administered AAV8. TAFA4 (test group), both A and B wave amplitudes recovered, particularly B wave amplitude, which returned to near-normal levels. Figure 3 ).

[0549] 2-C. Efficacy evaluation of interspecific TAFA4 variants in a NaIO3 (sodium iodate)-induced retinal injury model

[0550] To determine whether interspecific variants of TAFA4 also exhibit similar efficacy, sequence identity of the full-length TAFA4 protein from mammals (humans, monkeys, pigs, rabbits, rats, mice), birds (chickens), reptiles (komodo dragons, walllizards, fence lizards, geckos), amphibians (frogs), and fish was compared, and shared sequences among species were identified. Figure 4 The amino acid sequence (95 aa) of the mature human TAFA4 protein showed over 90% sequence identity compared to mammals and amphibians, over 95% compared to birds, and over 85% compared to reptiles and fish, confirming very high cross-species sequence identity. Figure 5 ).

[0551] Since the neuroprotective effect of AAV8. mouse TAFA4 on retinal injury was confirmed in a NaIO3-induced retinal injury model, additional experiments were conducted to determine whether TAFA4 from other species also possesses retinal protective effects. Specifically, we evaluated the efficacy of human TAFA4, as well as TAFA4 from reptiles (gecko TAFA4; sequence identity 87.4%) and fish (fish TAFA4; sequence identity 86.3%), which showed the lowest sequence identity with human TAFA4. Changes in fluorescence angiography (FA) and electroretinography (ERG) following administration of AAV8. human TAFA4, AAV8. gecko TAFA4, and AAV8. fish TAFA4 were assessed in a NaIO3-induced AMD mouse model.

[0552] C57BL / 6 mice (Orient Bio) were administered control AAV8 or species-derived AAV8.TAFA4 via subretinal injection (SRI).

[0553] To allow for adequate TAFA4 expression, 1×10⁻⁶ TAFA4 was injected subretinally into both eyes of mice. 9 After a maximum of 42 days, mice were injected with AAV8 via vg and NaIO3 was administered intravenously at a dose of 20 mg / kg to induce an AMD model. On day 9 post-induction (day 51 post-SRI), fluorescein dye was injected via the tail vein. Subsequently, fundus imaging and FA images were acquired using a Micron-IV imaging camera (Phoenix). On day 7 post-induction (day 49 post-SRI), dark-adapted ERG was performed. Mice were placed on an ERG stage, and ERG probes were placed in contact with the tail, head, and cornea. A-wave and B-wave amplitudes were then measured. ERG analysis was performed using the LabScribeERG (iWorx Data Acquisition Software) program.

[0554] On FA images taken on day 9 after model induction, leakage of fluorescein dye due to damage to the outer retina was observed in the NaIO3-treated group (control group, treated with control AAV8) compared to healthy animals (initial group). Conversely, the groups treated with AAV8.human TAFA4, AAV8.g.g.g., or AAV8.g.g.g., fish TAFA4) (test group) showed a nearly normal retinal appearance similar to the initial group. Figure 6Similarly, dark-adapted ERG assessed on day 11 post-model induction showed a significant reduction in induced A and B wave amplitudes due to NaIO3-induced retinal abnormalities. Conversely, in the groups treated with AAV8.human TAFA4, AAV8.gecko TAFA4, or AAV8.fish TAFA4 (test groups), NaIO3-induced A and B wave amplitude changes were recovered. Figure 7 ).

[0555] 2-D. Retinal protective effect of TAFA4 paralogs based on sequence identity in a NaIO3 (sodium iodate)-induced retinal injury model.

[0556] High sequence identity has been reported between TAFA4 and other TAFA family proteins (TAFA1 through TAFA3). Therefore, we examined whether human TAFA1, TAFA2, and TAFA3 also exhibit retinal protective effects in a NaIO3-induced mouse model of retinal injury. In a NaIO3-induced mouse AMD model, changes in FA and ERG following administration of AAV8.TAFA1, AAV8.TAFA2, AAV8.TAFA3, and AAV8.TAFA4 were evaluated.

[0557] Control AAV8, AAV8.TAFA1, AAV8.TAFA2, AAV8.TAFA3, or AAV8.TAFA4 were administered to C57BL / 6 mice (Orient Bio) via subretinal injection (SRI).

[0558] To evaluate the retinal protective effects of TAFA1, TAFA3, and TAFA4, 1×10⁻⁶ tbsp was injected subretinally into both eyes of mice. 9 After a maximum of 42 days, mice were injected with AAV8 via vg and NaIO3 was administered intravenously at a dose of 20 mg / kg to induce an AMD model. On day 9 post-induction (day 51 post-SRI), fluorescein dye was injected via the tail vein, and fundus images were visualized using a Micron-IV imaging camera (Phoenix) to acquire FA images. On day 7 post-induction (day 49 post-SRI), dark-adapted ERG was performed. Mice were placed on an ERG stage, and ERG probes were placed in contact with the tail, head, and cornea, and the amplitudes of A and B waves were measured. ERG analysis was performed using LabScribeERG (iWorx Data Acquisition Software).

[0559] To evaluate the retinal protective effect of TAFA2, 1×10⁻⁶ tbsp was injected subretinally into both eyes of mice. 9After a maximum wait of 56 days, AAV8 was administered intravenously at a dose of 20 mg / kg to induce an AMD model. On day 9 post-induction (day 65 post-SRI), fluorescein dye was injected via the tail vein, and fundus images were visualized using a Micron-IV imaging camera (Phoenix) to acquire FA images. On day 7 post-induction (day 63 post-SRI), dark-adapted ERG was performed, and A-wave and B-wave amplitudes were measured and analyzed as described above.

[0560] On FA images, leakage of fluorescein dye due to damage to the outer retina was observed in the NaIO3-treated groups (control group, AAV8-treated group) compared to healthy animals (initial group, AAV8-treated group). Conversely, the groups treated with AAV8.TAFA1, AAV8.TAFA2, AAV8.TAFA3, or AAV8.TAFA4 (test group) showed a nearly normal retinal appearance similar to the initial group. Figure 8 Dark adaptation ERG also revealed that NaIO3-induced A-wave and B-wave amplitudes were significantly reduced, indicating retinal abnormalities. Conversely, in the groups treated with AAV8.TAFA1, AAV8.TAFA2, AAV8.TAFA3, or AAV8.TAFA4 (test group), NaIO3-induced A-wave and B-wave amplitude changes were restored. Figure 9 ).

[0561] 2-E. Comparison of sequence identity among TAFA1 to TAFA4 variants across species

[0562] As confirmed in Sections 2-C and 2-D, retinal protection was observed in interspecific variants of TAFA4 and its paralogs TAFA1 through TAFA3 in a NaIO3-induced retinal injury model. Therefore, we hypothesized that shared sequences among various TAFA proteins might confer retinal protective activity, and we compared the sequence identity of full-length TAFA1, TAFA2, TAFA3, and TAFA4 proteins from vertebrates—mammals (human, monkey, pig, rabbit, rat, mouse), birds (chicken), reptiles (Komodo dragon, wall lizard, fence lizard, gecko), amphibians (frog), and fish—to identify shared sequences. The amino acid sequences of full-length TAFA1 through TAFA4 in vertebrates are shown below. Figures 10A to 10D .

[0563] The 93-amino acid sequence of human TAFA4 (IKQGTCEVVAVHRCCNKNRIEERSQTVKCSCFPGQVAGTTRAQPSCVEASIVIQKWWCHMNPCLEGEDCKVLPDYSGWSCSSGNKVKTTKVTR, SEQ ID NO: 87) shows very high sequence identity with TAFA4 sequences from other species and with shared sequences from TAFA1 to TAFA3 from humans to fish. Sequence identity within shared regions is summarized in Table 2.

[0564] [Table 2]

[0565] The sequence identity of the 93 common amino acids in human TAFA4 with those of TAFA1, TAFA2, TAFA3, and TAFA4 from various species.

[0566]

[0567]

[0568]

[0569] Clustal Omega was used to determine sequence identity.

[0570] [Example 3] Efficacy evaluation of TAFA4 peptide fragment

[0571] Based on the results of Examples 1 and 2, the inventors synthesized three TAFA4 peptide fragments (TAFA4 fragments 1 to 3, F1 to F3) as shown in Table 3 to identify the TAFA4 fragments exhibiting biological activity. The peptides were synthesized by Abclon.

[0572] [Table 3]

[0573] Amino acid sequences of TAFA4 peptide fragments 1 to 3

[0574]

[0575] As in Example 1, MED17.11 cells resuspended in differentiation medium were at a concentration of 1 × 10⁻⁶. 4Cells / mL, 100 μL per well, were seeded in 96-well cell culture plates. TAFA4 peptide fragments 1 through 3 were added at 5 μM, and an equal volume of dimethyl sulfoxide (DMSO, Sigma) was added to the control group. The plates were then incubated at 37°C in a 5% CO2 incubator. After 4 days of culture, neurite length and branching point number were analyzed using the Incucyte system. Three independent experiments were performed, and an increasing trend in neurite length and branching point number was observed in the groups treated with TAFA4 fragment 2 (F2) and TAFA4 fragment 3 (F3). Figure 11A and Figure 11B ).

[0576] The interspecific sequences of TAFA4 fragment 2 (F2) and fragment 3 (F3) are shown in Tables 4 and 5, respectively.

[0577] [Table 4]

[0578] Interspecific amino acid sequence of TAFA4 fragment 2

[0579]

[0580] The sequence reflecting fragment 2 and its interspecific variants corresponds to SEQ ID NO: 147.

[0581] [Table 5]

[0582] Interspecific amino acid sequence of TAFA4 fragment 3

[0583]

[0584] The sequence reflecting fragment 3 and its interspecific variants corresponds to SEQ ID NO: 144.

[0585] [Example 4] Efficacy evaluation of subdivided TAFA4 peptide fragments

[0586] Based on Example 3, where TAFA4 fragments 2 (F2) and 3 (F3) exhibited activity, further subdivided TAFA4 peptide fragments (TAFA4 fragments 4 to 8, F4 to F8) were synthesized as shown in Table 6 to identify the smallest TAFA4 peptide fragments exhibiting biological activity. The peptides were synthesized by Abclon, and the positions of each fragment are shown in Table 6. Figure 12 middle.

[0587] [Table 6]

[0588] Amino acid sequence of TAFA4 peptide fragments 4 to 8

[0589]

[0590] Using the same experimental procedure as in Example 4, 5 μM of each TAFA4 peptide fragment or an equal volume of DMSO was added, and the cells were incubated at 37°C (5% CO2). After 4 days, cell images were acquired using Incucyte (Sartorius), and neurite length and branching points were analyzed. Two independent experiments were performed, and statistical significance was assessed by one-way ANOVA with Dunnett's post-hoc test.

[0591] The neurite lengths of TAFA4 fragments 5 (F5) and 7 (F7) tend to increase, with fragment 5 (F5) showing a statistically significant increase. Figure 13A Similarly, only TAFA4 fragments 5 (F5) and 7 (F7) showed a trend of increasing branch point numbers. Figure 13B ).

[0592] Interspecific sequence homology of TAFA4 fragment 5 (F5) and fragment 7 (F7) is shown in Tables 7 and 8.

[0593] [Table 7]

[0594] Interspecific sequence identity comparison of TAFA4 peptide fragment 5

[0595]

[0596] [Table 8]

[0597] Interspecific sequence identity comparison of TAFA4 peptide fragment 7

[0598]

[0599] [Example 5] Evaluation of the efficacy of TAFA4 and its fragments in vivo for neuropathic pain

[0600] As described above, rhTAFA4, along with TAFA4 fragments 2, 3, 5, and 7 (F2, F3, F5, and F7), increased neurite length and the number of branch points in MED17.11 cells. Based on this, we evaluated the efficacy of rhTAFA4 and TAFA4 fragments in an in vivo neuropathic pain model.

[0601] Six-week-old mice were anesthetized, and the skin between the gluteus maximus and biceps femoris muscles in the left hind limb was incised to expose the sciatic nerve. After separating the exposed nerve from the surrounding tissue, the proximal portions of the nerve, where it bifurcates into the tibial, peroneal, and sural nerves, were ligated three times at intervals of 0.5 mm to 1 mm using 6-0 silk sutures (AILEE). The skin was sutured, and the animals were allowed to recover for one week. One week later, pain was assessed using the von Frey test. In the von Frey test, starting with the minimum force (0.008 g), each monofilament was applied six times to the center of the plantar surface of the left hind paw. If no pain response (lifting, shaking, licking, etc.) was observed, monofilaments with progressively increasing force were used. The g-values ​​of the monofilaments that elicited a pain response at least three times out of the six applications were recorded, and the 50% threshold was calculated.

[0602] After confirming pain induced by sciatic nerve ligation, rhTAFA4 and TAFA4 fragments were administered to assess pain relief. rhTAFA4 and TAFA4 fragments 1, 2, 3, 5, 6, and 7 (F1, F2, F3, F5, F6, and F7) were diluted to 200 μg / mL in PBS and administered intrathecally at the L4-L5 level, 10 μL (2 μg) per mouse. One hour after administration, the von Frey test was repeated to assess pain relief.

[0603] In the control group receiving the same volume of PBS, pain induced by nerve ligation persisted, indicating the presence of mechanical aberrant pain. Conversely, pain was relieved in the rhTAFA4-treated group. Pain also persisted in the groups treated with TAFA4 fragments 1 and 6 (F1 and F6), while pain was relieved in the groups treated with TAFA4 fragments 2, 3, 5, and 7 (F2, F3, F5, and F7), which have shown a significant tendency to increase neurite length and branching points. Figure 14 ).

[0604] Fragments 2 and 5, which exhibit efficacy, share the sequence GQVAGTTRAQPSCVEAS (17 amino acids, referred to as fragment 2.5), and fragments 3 and 7 share the sequence EGEDCKVL (8 amino acids, referred to as fragment 3.7). Therefore, the increased neurite length and branching points observed in Examples 1 to 4, and the analgesic effect observed in this example, are considered to be attributable to the polypeptide containing the shared sequence of fragment 2.5 or fragment 3.7.

[0605] Comparison of the sequence of TAFA4 fragment 2.5 with the interspecific variant sequences of TAFA1 to TAFA3 revealed that the human TAFA4 fragment 2.5 differs from the human fragment by only one to four amino acids, indicating very high homology.

[0606] Similarly, comparing the sequence of TAFA4 fragment 3.7 with the interspecific variant sequences of TAFA1 to TAFA3 showed that the human TAFA4 fragment 3.7 differs from the human fragment by only one to three amino acids, confirming that fragment 3.7 also has very high homology.

[0607] The results of comparing the TAFA4 fragment with interspecific variant sequences of TAFA1 to TAFA3 are summarized in Table 9 (fragment 2.5) and Table 10 (fragment 3.7).

[0608] [Table 9]

[0609] The sequences of TAFA protein fragment 2.5 and its interspecific variants that exhibit efficacy.

[0610]

[0611]

[0612]

[0613] Differences in amino acids are shown in bold. The sequence of fragment 2.5, including its interspecies variants, corresponds to SEQ ID NO:145.

[0614] [Table 10]

[0615] The sequences of TAFA protein fragment 3.7 and its interspecific variants that exhibit efficacy.

[0616]

[0617]

[0618]

[0619] Differences in amino acids are shown in bold. The sequence of fragment 3.7, including its interspecies variants, corresponds to SEQ ID NO:142.

[0620] [Example 6] Homology comparison between TAFA4 peptide fragment and TAFA1 to TAFA3 peptide fragments

[0621] In Example 3, we confirmed that the interspecies homology of the TAFA4 fragment was at least 70% (≥81.3% for F5 and ≥72.7% for F7). High sequence identity has been reported between TAFA4 and other TAFA family proteins. Therefore, we hypothesized that shared sequences among various TAFA peptide fragments might confer the effect of increasing neurite length and branching points, and we compared the sequence homology between TAFA1, TAFA2, and TAFA3 peptide fragments from vertebrates—mammals (human, monkey, pig, rabbit, rat, mouse), birds (chicken), reptiles (Komodo dragon, wall lizard, fence lizard, gecko), amphibians (frog), and fish—and human TAFA4 fragments 5 (F5) and 7 (F7), summarized in Tables 11 to 16, to identify shared sequences.

[0622] [Table 11]

[0623] Sequence identity comparison between human TAFA4 fragment 5 and TAFA1 fragment 5

[0624]

[0625] [Table 12]

[0626] Sequence identity comparison between human TAFA4 fragment 5 and TAFA2 fragment 5

[0627]

[0628] [Table 13]

[0629] Sequence identity comparison between human TAFA4 fragment 5 and TAFA3 fragment 5

[0630]

[0631] The sequence of fragment 5, including its interspecific variants, corresponds to SEQ ID NO: 146.

[0632] [Table 14]

[0633] Sequence identity comparison between human TAFA4 fragment 7 and TAFA1 fragment 7

[0634]

[0635] [Table 15]

[0636] Sequence identity comparison between human TAFA4 fragment 7 and TAFA2 fragment 7

[0637]

[0638] [Table 16]

[0639] Sequence identity comparison between human TAFA4 fragment 7 and TAFA3 fragment 7

[0640]

[0641] The sequence of fragment 7, including its interspecific variants, corresponds to SEQ ID NO: 143.

[0642] In addition, the interspecific sequences of fragment 3 from TAFA1 to TAFA3 (Table 17) and the interspecific sequences of fragment 2 from TAFA1 to TAFA3 (Table 18) are as follows:

[0643] [Table 17]

[0644] Interspecific sequences of fragments 3 from TAFA1 to TAFA3

[0645]

[0646]

[0647] [Table 18]

[0648] Interspecific sequences of fragments 2 from TAFA1 to TAFA3

[0649]

[0650]

[0651] All publications, patents, patent applications and other documents cited herein are incorporated herein by reference in their entirety for all purposes, regardless of whether each individual publication, patent, patent application or other document is expressly stated to be incorporated herein by reference for all purposes.

[0652] Although this disclosure has been described with reference to the foregoing embodiments, those skilled in the art will understand that various modifications and changes can be made, such as adding, altering, deleting, or inserting components, without departing from the spirit of this disclosure as set forth in the claims. Such modifications and changes should be understood to fall within the scope of this disclosure.

Claims

1. A polypeptide having the ability to increase the length of neurites or the number of branch points, wherein the polypeptide: (i) Composed of 8 to 61 amino acid residues; and (ii) A sequence having at least 50% sequence identity with the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO:

15.

2. The polypeptide of claim 1, wherein the amino acid sequence of the polypeptide from the N-terminus to the C-terminus comprises the amino acid sequence of general formula 1: General Formula 1: In general formula 1, X1 can be E, D, P, L, or A. X2 is either D or E, and X3 can be T, V, I, or A.

3. The polypeptide according to claim 2, wherein the amino acid sequence of the polypeptide comprises the amino acid sequence of SEQ ID NO:

142.

4. The polypeptide according to claim 2, wherein the amino acid sequence of the polypeptide comprises an amino acid sequence selected from SEQ ID NO: 1 to SEQ ID NO:

14.

5. The polypeptide according to claim 2, wherein the amino acid sequence of the polypeptide consists of 8 to 43 amino acid residues.

6. The polypeptide of claim 1, wherein the amino acid sequence of the polypeptide from the N-terminus to the C-terminus comprises the amino acid sequence of general formula 2: General Formula 2: In general formula 2, X1 can be E, D, P, L, or A. X2 is either D or E. X3 can be T, V, I, or A. X4 can be I, E, A, L, or V. X5 is Q, E, or G. X6 is either K or R. X7 is E, H, or Q. X8 is E, Q, N, D, S, or H, and X9 can be L, M, or V.

7. The polypeptide according to claim 6, wherein the amino acid sequence of the polypeptide comprises the amino acid sequence of SEQ ID NO:

143.

8. The polypeptide of claim 6, wherein the amino acid sequence of the polypeptide comprises an amino acid sequence selected from SEQ ID NO: 28 to SEQ ID NO:

51.

9. The polypeptide of claim 1, wherein the amino acid sequence of the polypeptide from the N-terminus to the C-terminus comprises the amino acid sequence of general formula 3: General Formula 3: In general formula 3, X1 can be E, D, P, L, or A. X2 is either D or E. X3 can be T, V, I, or A. X4 can be Y, S, or L. X5 is either S or T. X6 is either S or T, and X7 is either V or I.

10. The polypeptide of claim 9, wherein the amino acid sequence of the polypeptide comprises the amino acid sequence of SEQ ID NO:

144.

11. The polypeptide of claim 9, wherein the amino acid sequence of the polypeptide comprises an amino acid sequence selected from SEQ ID NO: 52 to SEQ ID NO:

58.

12. The polypeptide of claim 1, wherein the amino acid sequence of the polypeptide from the N-terminus to the C-terminus comprises the amino acid sequence of general formula 4: Formula 4: In general formula 4, X1 is either Q or K. X2 is R, H, or Q. X3 is A, N, S, or T. X4 can be R, A, Q, K, or T. X5 is either D or E. X6 is either A or does not exist, and X7 is S, A, or does not exist.

13. The polypeptide of claim 12, wherein the amino acid sequence of the polypeptide comprises the amino acid sequence of SEQ ID NO:

145.

14. The polypeptide of claim 12, wherein the amino acid sequence of the polypeptide comprises an amino acid sequence selected from SEQ ID NO: 15 to SEQ ID NO:

27.

15. The polypeptide of claim 12, wherein the amino acid sequence of the polypeptide consists of 15 to 46 amino acid residues.

16. The polypeptide of claim 1, wherein the amino acid sequence of the polypeptide from the N-terminus to the C-terminus comprises the amino acid sequence of general formula 5: Formula 5: In general formula 5, X1 is either Q or K. X2 is R, H, or Q. X3 is A, N, S, or T. X4 can be R, A, Q, K, or T. X5 is either D or E. X6 is either A or does not exist. X7 is S, A, or does not exist. X8 is either R or K. X9 is either R or L. X10 is either V or G. X11 is either K or N. X12 is F or L, and X13 is either P or S.

17. The polypeptide of claim 16, wherein the amino acid sequence of the polypeptide comprises the amino acid sequence of SEQ ID NO:

146.

18. The polypeptide of claim 16, wherein the amino acid sequence of the polypeptide comprises an amino acid sequence selected from SEQ ID NO: 59 to SEQ ID NO:

74.

19. The polypeptide of claim 1, wherein the amino acid sequence of the polypeptide from the N-terminus to the C-terminus comprises the amino acid sequence of general formula 6: Formula 6: In general formula 6, X1 is either Q or K. X2 is R, H, or Q. X3 is A, N, S, or T. X4 can be R, A, Q, K, or T. X5 is either D or E. X6 is either A or does not exist. X7 is S, A, or does not exist. X8 can be I, A, V, or L. X9 is either Q or E. X10 is either H or Q. X11 is N, D, S, or H, and X12 is either L or M.

20. The polypeptide of claim 19, wherein the amino acid sequence of the polypeptide comprises the amino acid sequence of SEQ ID NO:

147.

21. The polypeptide of claim 19, wherein the amino acid sequence of the polypeptide comprises an amino acid sequence selected from SEQ ID NO: 75 to SEQ ID NO:

85.

22. The polypeptide of claim 1, wherein the amino acid sequence of the polypeptide comprises an amino acid sequence selected from SEQ ID NO: 152 to SEQ ID NO:

184.

23. A nucleic acid molecule encoding a polypeptide according to any one of claims 1 to 22.

24. A carrier comprising the nucleic acid molecule according to claim 23.

25. A recombinant viral particle comprising the vector and capsid protein as described in claim 24.

26. The recombinant viral particle of claim 25, wherein the virus is AAV.

27. A cell comprising the carrier according to claim 24.

28. A cell transformed using the vector according to claim 24.

29. A composition comprising a polypeptide according to any one of claims 1 to 22, a nucleic acid molecule encoding the polypeptide, a vector comprising the nucleic acid molecule, a recombinant viral particle comprising the vector and a capsid protein, a cell comprising the vector, a cell transformed with the vector, or any combination thereof.

30. The composition according to claim 29, wherein the composition is a pharmaceutical composition.

31. The composition according to claim 30, wherein the pharmaceutical composition is a composition for the prevention or treatment of retinal neurodegenerative diseases.

32. The composition of claim 31, wherein the retinal neurodegenerative disease comprises retinopathy, choroidal neovascularization, macular degeneration, retinal degeneration, macular edema, retinal swelling, macular swelling, retinal swelling, color vision deficiency, retinal cell degeneration, retinal vascular occlusion, retinal detachment, hereditary retinal disease, or any combination thereof.

33. The composition of claim 30, wherein the pharmaceutical composition is a composition for the prevention or treatment of neuropathic pain.

34. The composition of claim 33, wherein the neuropathic pain is abnormal pain, hyperalgesia, hyperesthesia, or sensory disturbance.

35. The composition of claim 33, wherein the neuropathic pain is central or peripheral neuropathic pain.

36. A method for producing a composition comprising the step of preparing the composition, said composition comprising a polypeptide according to any one of claims 1 to 22, a nucleic acid molecule encoding said polypeptide, a vector comprising said nucleic acid molecule, a recombinant viral particle comprising said vector and a capsid protein, a cell comprising said vector, a cell transformed with said vector, or any combination thereof.

37. A therapeutic use for preparing a medicament comprising a polypeptide according to any one of claims 1 to 22, a nucleic acid molecule encoding the polypeptide, a carrier comprising the nucleic acid molecule, a recombinant viral particle comprising the carrier and a capsid protein, a cell comprising the carrier, a cell transformed with the carrier, or any combination thereof.

38. A method for preventing or treating a disease or ailment in a subject in need, comprising administering a composition to the subject, the composition comprising a polypeptide according to any one of claims 1 to 22, a nucleic acid molecule encoding the polypeptide, a vector comprising the nucleic acid molecule, a recombinant viral particle comprising the vector and a capsid protein, a cell comprising the vector, a cell transformed with the vector, or any combination thereof.

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