Composition for treating inner ear diseases

Abstract

The present invention relates to a polypeptide that induces ribbon synapse formation in the inner ear or increases the number of ribbon synapses in the inner ear, or improves the function of ribbon synapses. The present invention also relates to a polypeptide having the ability to promote neurite outgrowth or increase branch points of spiral ganglion neurons. A pharmaceutical composition comprising a polypeptide, a nucleic acid molecule encoding the polypeptide, a vector comprising the nucleic acid molecule, recombinant viral particles comprising the vector and a capsid protein, a cell comprising the vector, a cell transformed with the vector, or a combination thereof, of the present invention, prevents or ameliorates damage to or abnormality of the auditory system or the equilibrium system (vestibular system), and thus can be effectively used as a therapeutic agent for inner ear diseases.

Description

Composition for the treatment of inner ear diseases

[0001] The present invention relates to a polypeptide comprising an amino acid sequence of a TAFA protein or a fragment thereof or a variant thereof, 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, or a cell transformed with said vector. Furthermore, the present invention relates to a composition for treating inner ear diseases and / or diseases caused by damage or abnormalities of inner ear ribbon synapses comprising said polypeptide, said nucleic acid molecule encoding said polypeptide, a vector comprising said nucleic acid molecule, said recombinant viral particle comprising said vector and a capsid protein, said vector, or a cell transformed with said vector, or a combination thereof.

[0002]

[0003] The TAFA protein family is a group of proteins that are abundantly expressed throughout brain regions and consists of five proteins, from TAFA1 to TAFA5. These proteins contain structurally conserved cysteine ​​residues, and the C, CC, and CXC motifs between the cysteines are associated with chemokines.

[0004] These proteins have been shown to be evolutionarily well-conserved within vertebrates and important for the normal functioning of the central nervous system. In mice with TAFA1 knockout, reduced body weight, decreased anxiety behavior, and impaired memory regarding fear have been reported (Lei X, Liu L, Terrillion CE, et al. FASEB J. 2019;33(12):14734-14747. and Yong HJ, Ha N, Cho EB, et al. Sci Rep. 2020;10(1):3969.). Conversely, an increase in anxious behavior has been reported when TAFA2 and TAFA3 are knocked out (Choi JH, Jeong YM, Kim S, et al. Proc Natl Acad Sci US A. 2018;115(5):E1041-E1050. and Kim S, Lee B, Choi JH, Kim JH, Kim CH, Shin HS. Sci Rep. 2017;7(1):16503.). Allodynia and hyperalgesia have been reported in TAFA4-null mice (Delfini MC, Mantilleri A, Gaillard S, et al. Cell Rep. 2013;5(2):378-388.). Behavioral changes, such as increased depressive behavior and loss of spatial memory ability, have been reported in mice with TAFA5 knockout (Huang S, Zheng C, Xie G, et al. FAM19A5 / TAFA5, a novel neurokine, plays a crucial role in depressive-like and spatial memory-related behaviors in mice. Mol Psychiatry. 2021;26(6):2363-2379.)

[0005] Despite the important roles these proteins play in the central nervous system, what is known about the functions of each individual protein, as well as their pathophysiological or therapeutic functions, is very limited, and their roles outside the central nervous system have also been elucidated to a limited extent.

[0006]

[0007] The matters described as background technology above are intended only to enhance understanding of the background of the present invention and should not be construed as an acknowledgment that they constitute prior art already known to those skilled in the art.

[0008]

[0009] The object of the present invention is to provide a polypeptide comprising an amino acid sequence of a TAFA (TAFA Chemokine Like Family Members) protein or a fragment thereof or a variant thereof, a nucleic acid molecule encoding said polypeptide, a vector comprising said nucleic acid molecule, a recombinant virus particle comprising said vector and a capsid protein, a cell comprising said vector, or a cell transformed with said vector.

[0010] Another object of the present invention is to provide a pharmaceutical composition for the prevention or treatment of inner ear disease comprising (i) a polypeptide comprising an amino acid sequence of a TAFA (TAFA Chemokine Like Family Members) protein or a fragment thereof or a variant thereof, a nucleic acid molecule encoding said polypeptide, a vector comprising said nucleic acid molecule, a recombinant virus particle comprising said vector and a capsid protein, a cell comprising said vector, a cell transformed with said vector, or a combination thereof, and (ii) a pharmaceutically acceptable carrier.

[0011] Another object of the present invention is to provide a pharmaceutical composition for the prevention or treatment of a disease caused by damage or abnormality of ribbon synapses in the inner ear, comprising: (i) a polypeptide comprising an amino acid sequence of a TAFA (TAFA Chemokine Like Family Members) protein or a fragment thereof or a variant thereof; a nucleic acid molecule encoding said polypeptide; a vector comprising said nucleic acid molecule; a recombinant virus particle comprising said vector and a capsid protein; a cell comprising said vector; a cell transformed with said vector; or a combination thereof; and (ii) a pharmaceutically acceptable carrier.

[0012] Another objective of the present invention is to provide a method for preparing a pharmaceutical composition for the prevention or treatment of inner ear disease, comprising the step of preparing a composition comprising a polypeptide having an amino acid sequence of the TAFA protein or a fragment thereof or a variant thereof, a nucleic acid molecule encoding the polypeptide, a vector having the nucleic acid molecule, a recombinant virus particle having the vector and a capsid protein, a cell having the vector, a cell transformed with the vector, or a combination thereof.

[0013] Another objective of the present invention is to provide a method for preparing a pharmaceutical composition for the prevention or treatment of a disease caused by damage or abnormality of ribbon synapses in the inner ear, comprising the step of preparing a composition comprising a polypeptide having an amino acid sequence of the TAFA protein or a fragment thereof or a variant thereof, a nucleic acid molecule encoding the polypeptide, a vector having the nucleic acid molecule, a recombinant virus particle having the vector and a capsid protein, a cell having the vector, a cell transformed with the vector, or a combination thereof.

[0014] Another objective of the present invention is to provide a use for the prevention or treatment of inner ear disease for the manufacture of a drug comprising a polypeptide comprising an amino acid sequence of the TAFA protein or a fragment thereof or a variant thereof, a nucleic acid molecule encoding the polypeptide, a vector comprising the nucleic acid molecule, a recombinant virus particle comprising the vector and a capsid protein, a cell comprising the vector, a cell transformed with the vector, or a combination thereof.

[0015] Another objective of the present invention is to provide a drug for the prevention or treatment of diseases caused by inner ear ribbon synapse damage, comprising a polypeptide comprising the amino acid sequence of the TAFA protein or a fragment thereof or a variant thereof, a nucleic acid molecule encoding the polypeptide, a vector comprising the nucleic acid molecule, a recombinant virus particle comprising the vector and a capsid protein, a cell comprising the vector, a cell transformed with the vector, or a combination thereof.

[0016] Another objective of the present invention is to provide a method for preventing or treating an inner ear disease in a subject in need, comprising administering to the subject a composition comprising a polypeptide having an amino acid sequence of the TAFA protein or a fragment thereof or a variant thereof, a nucleic acid molecule encoding the polypeptide, a vector having the nucleic acid molecule, a recombinant virus particle having the vector and a capsid protein, a cell having the vector, a cell transformed with the vector, or a combination thereof.

[0017] Another objective of the present invention is to provide a method for preventing or treating a disease caused by damage to ribbon synapses in the inner ear in a subject in need, comprising administering to the subject a composition comprising a polypeptide having an amino acid sequence of the TAFA protein or a fragment thereof or a variant thereof, a nucleic acid molecule encoding the polypeptide, a vector having the nucleic acid molecule, a recombinant virus particle having the vector and a capsid protein, a cell having the vector, a cell transformed with the vector, or a combination thereof.

[0018]

[0019] Other objects and advantages of the present invention will become more apparent from the following detailed description of the invention, claims, and drawings.

[0020]

[0021] The present invention generally relates to compositions for treating inner ear diseases. More specifically, the present invention presents for the first time that hearing can be improved by administering a polypeptide comprising an amino acid sequence of a TAFA protein, a fragment thereof, or a variant thereof, a nucleic acid molecule encoding said polypeptide, or a vector comprising said nucleic acid molecule. As described further herein, the polypeptide provided by the present invention may be used to reduce damage to ribbon synapses within the inner ear of a subject (e.g., a subject suffering from an inner ear disease) and / or to induce the formation of ribbon synapses within the inner ear of the subject. Additional aspects are provided throughout the present invention.

[0022] Numerous terms and phrases are defined to facilitate understanding of the contents disclosed in this specification. Additional definitions are provided throughout the detailed description.

[0023]

[0024] I. Definition

[0025] As used herein, the term “TAFA protein fragment” means a fragment of TAFA1 to TAFA4 proteins comprising a minimum size fragment capable of reducing the growth of neurites and / or damage to ribbon synapses in the inner ear or inducing the formation of ribbon synapses. The above fragments are 8 (e.g., "TAFA protein fragment 3.7"), 9, 10, 11, 12, 13, 14, 15, 16, 17 (e.g., "TAFA protein fragment 2.5"), 18, 19, 20, 21, 22 (e.g., "TAFA protein fragment 7"), 23, 24, 25 (e.g., "TAFA protein fragment 2.5" + "TAFA protein fragment 3.7"), 26, 27, 28, 29 (e.g., "TAFA protein fragment 3"), 30, 31 (e.g., "TAFA protein fragment 2"), 32 (e.g., "TAFA protein fragment 5"), 33, 34, 35, 36, 37, It may consist of, but is not limited to, a sequence of 38, 39 (e.g., "TAFA protein fragment 2.5"+"TAFA protein fragment 7"; e.g., "TAFA protein fragment 2"+"TAFA protein fragment 3.7"), 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54 (e.g., "TAFA protein fragment 5"+"TAFA protein fragment 7"), 55, 56, 57, 58, 59, 60 (e.g., "TAFA protein fragment 2"+"TAFA protein fragment 3") or 61 (e.g., "TAFA protein fragment 5"+"TAFA protein fragment 3") amino acids.

[0026] As used herein, the term "variant of a TAFA protein fragment" includes cases where one or more amino acids of the polypeptide constituting the TAFA1 to TAFA4 protein fragments are modified by substitution, deletion, or insertion, and variants in which the biological activity or function of the polypeptide is substantially maintained despite such modification are also included within the scope of the present invention. The variant is preferably a functional variant. The term "functional" refers to a variant derived from the TAFA1 to TAFA4 protein fragments having the ability to reduce the growth of neurites and / or damage to ribbon synapses in the inner ear, or to induce the formation of ribbon synapses, similar to the "TAFA protein fragment." The variant is preferably an interspecies variant (e.g., a polypeptide comprising any one of the amino acid sequences of SEQ ID NOs 142 to 147). A person skilled in the art can easily produce polypeptides by utilizing the difference in non-conserved sequences, excluding conserved sequences, among the amino acid sequences of the known TAFA1 to TAFA4 protein fragments of each species, to substitute one or more amino acids with other amino acids (for example, by substituting the 4th sequence D with E by referring to the 3.7 sequence of the human TAFA4 protein fragment EGEDCKVL (Sequence No. 1) with the 4th sequence D by referring to the 3.7 sequence of the rabbit TAFA2 protein fragment EGEECKVL (Sequence No. 6), or by substituting the 1st sequence E with P and / or substituting the 4th sequence D with E by referring to the 3.7 sequence of the porcine TAFA3 protein fragment PGEECKVL (Sequence No. 9).

[0027] The above amino acid substitution may be a substitution of 1 to 22 amino acids. For example, if the TAFA protein fragment contains fragments 5 and 7 simultaneously, 1 to 22 amino acid substitutions are possible; if the TAFA protein fragment contains fragments 2 and 3 simultaneously, 1 to 19 amino acid substitutions are possible; if the TAFA protein fragment contains only fragment 2, 1 to 12 amino acid substitutions are possible; if the TAFA protein fragment contains only fragment 5, 1 to 13 amino acid substitutions are possible; if the TAFA protein fragment contains only fragment 3, 1 to 7 amino acid substitutions are possible; if the TAFA protein fragment contains only fragment 7, 1 to 9 amino acid substitutions are possible; if the TAFA protein fragment contains only fragment 2.5, 1 to 7 amino acid substitutions are possible; and if the TAFA protein fragment contains only fragment 3.7, 1 to 3 amino acid substitutions are possible.

[0028] Preferably, the substitutions of the amino acids may be 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.

[0029] As used herein, the term "adeno-associated virus" (AAV) refers to a single-stranded DNA virus that is a helper-dependent human parvovirus. The genome size is approximately 4.6 kbp, and the N-terminal portion of the genome codes for the rep gene, which is involved in viral replication and the expression of viral genes, and the C-terminal portion codes for the cap gene, which encodes the viral capsid protein, and consists of repeat regions (ITRs) of approximately 145 bases inserted at both ends. The function of the 145 bp ITRs (inverted terminal repeats), which have a T-shaped structure, is to serve as the replication origin during viral genome replication and to act as a primary packaging signal. ITR is the only cis-acting sequence required when constructing a recombinant AAV construct, and it has enhancer activity in the presence of the Rep protein but very weak activity in the absence of the Rep protein. Therefore, when cloning foreign genes into a recombinant AAV construct, the enhancer, promoter, pA, etc. are appropriately configured to construct the expression construct, taking this into consideration (RJ Samulski and N Muzyczka, Annu. Rev. Virolo. 2014. 1:427-451). Four proteins are translated from the rep gene, which are classified as rep78, rep68, rep52, and rep40 according to their molecular weight and perform important functions in AAV DNA replication. Four proteins are translated from the cap gene, among which VP1, VP2, and VP3 proteins are structural proteins that constitute the AAV particle, and assembly-activating protein (AAP) promotes the assembly of the AAV particle by the said structural proteins.In order for the above adeno-associated virus to replicate efficiently, it requires some proteins and RNAs derived from helper viruses such as adenoviruses or herpes simplex viruses (Muzyczka N. Curr Top Microbiol Immunol 158, 97-129, 1992).

[0030] AAV includes, but is not limited to, AAV type 1, AAV type 2, AAV type 3 (including types 3A and 3B), AAV type 4, AAV type 5, AAV type 6, AAV type 7, AAV type 8, AAV type 9, AAV type 10, AAV type 11, AAV type 12, AAV type 13, AAVrh.74, snake AAV, avian AAV, bovine AAV, canine AAV, horse AAV, sheep AAV, goat AAV, shrimp AAV, AAV serotypes and lineage branches disclosed by Gao et al. (J. Virol. 78:6381 (2004)) and Moris et al. (Virol. 33:375 (2004)), and other AAVs currently known or that may be discovered in the future. For example, FIELDS et al. See VIROLOGY, volume 2, chapter 69 (4th ed., Lippincott-Raven Publishers). In some embodiments, "AAV" includes derivatives of known AAVs. In some embodiments, "AAV" includes modified or artificial AAVs. In some embodiments, "AAV" includes AAVs with modified capsids.

[0031] As used herein, the term “amino acid” includes 20 standard amino acids that naturally incorporate 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), cysteine ​​(C), etc.), as well as D-isomers and modified amino acids. Additionally, the peptide may include non-standard amino acids that have undergone post-translational modification. Post-translational modifications may include, but are not limited to, phosphorylation, glycosylation, acylation (e.g., acetylation, myristoylation, and palmitoylation), alkylation, carboxylation, hydroxylation, glycation, biotinylation, ubiquitinylation, changes in chemical properties (e.g., beta-removal deimidation, deamidation), and structural changes (e.g., formation of disulfide bridges). The peptide may be a wild-type peptide identified and isolated from a natural source. Meanwhile, the peptide may be an artificial variant comprising an amino acid sequence in which one or more amino acids are substituted, deleted, and / or inserted. Amino acid changes in wild-type polypeptides as well as artificial variants include conservative amino acid substitutions that do not significantly affect protein folding and / or activity.For example, the above-mentioned conservative substitutions may include basic amino acids (arginine (R), lysine (K), and histidine (H)), acidic amino acids (glutamic acid (E) and aspartic acid (D)), polar amino acids (glutamine (Q) and asparagine (N)), hydrophobic amino acids (leucine (L), isoleucine (I), valine (V), and methionine (M)), aromatic amino acids (phenylalanine (F), tryptophan (W), and tyrosine (Y)), and small amino acids (glycine (G), alanine (A), serine (S), and threonine (T)). In general, amino acid substitutions that do not alter specific activity are known in the art. The most common exchanges may include Ala / Ser, Val / Ile, Asp / Glu, Thr / Ser, Ala / Gly, Ala / Thr, Ser / Asn, Ala / Val, Ser / Gly, Tyr / Phe, Ala / Pro, Lys / Arg, Asp / Asn, Leu / Ile, Leu / Val, Ala / Glu, and Asp / Gly.

[0032] In some embodiments, if two or more sequences are 100% identical to each other, they are said to be "completely conserved" or "identical." In some embodiments, if two or more sequences are at least 70% identical, at least 80% identical, at least 90% identical, or at least 95% identical to each other, they are said to be "highly conserved." In some embodiments, if two or more sequences are approximately 70% identical, approximately 80% identical, approximately 90% identical, approximately 95%, approximately 98%, or approximately 99% identical to each other, they are said to be "highly conserved." In some embodiments, if two or more sequences are at least 30% identical, at least 40% identical, at least 50% identical, at least 60% identical, at least 70% identical, at least 80% identical, at least 90% identical, or at least 95% identical to each other, they are said to be "conserved." In some embodiments, two or more sequences are said to be "conserved" if they are about 30% identical, about 40% identical, about 50% identical, about 60% identical, about 70% identical, about 80% identical, about 90% identical, about 95% identical, about 98% identical, or about 99% identical to each other. Conservation of sequences may apply to the entire length of a polynucleotide or polypeptide, or to a part, region, or feature thereof.

[0033] The terms “complementary” and “complementaryness” refer to two or more oligomers (i.e., each containing a base sequence) or between an oligomer and a target gene that are associated with each other by Watson-Crick base pairing rules. For example, the base sequence “TGA(5’→3’) is complementary to the base sequence “ACT(3’→5’).” According to base pairing rules, if fewer than all bases of a given base sequence match another base sequence, the complementaryness may be “partial.” For example, in some embodiments, the complementaryness between a given base sequence and another base sequence may be about 70%, about 75%, about 80%, about 85%, about 90%, or about 95%. Accordingly, in certain embodiments, the term "complementary" refers to at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% agreement or complementarity with the target nucleic acid sequence. Or, for the sake of continuation, there may be "complete" or "perfect (100%)" complementarity between a given base sequence and another base sequence. In some embodiments, the degree of complementarity between base sequences has a significant effect on the efficiency and strength of hybridization between sequences.

[0034] The term "downstream" refers to a nucleotide sequence located at the 3' position relative to a reference nucleotide sequence. In certain embodiments, the downstream nucleotide sequence is related to a sequence after the starting point of transcription. For example, the translation start codon of a gene is located downstream of the transcription start site.

[0035] As used herein, the term “enhancer” refers to a portion of DNA containing a sequence capable of providing enhanced transcription and, in some cases, acting independently of its orientation toward another regulatory sequence. The enhancer may act in cooperation with or additionally with a promoter and / or other enhancer elements.

[0036] The terms “excipient” and “carrier” are used interchangeably and refer to an inert substance added to a pharmaceutical composition to further facilitate the administration of a compound, for example, a polynucleotide containing foreign genes and / or non-translating nucleic acid sequences as described herein.

[0037] The term "exon" refers to a nucleic acid sequence that appears in the mature form of an RNA molecule after a specific portion of a protein-coding nucleic acid or a portion of a pre-treated (or precursor) RNA has been removed by splicing. The mature RNA molecule may be messenger RNA (mRNA) or a functional form of non-coding RNA such as rRNA or tRNA.

[0038] As used herein, the term “expression” refers to the process by which a polynucleotide produces a gene product, e.g., RNA or polypeptide. This includes, but is not limited to, transcribing a polynucleotide into messenger RNA (mRNA) and translating mRNA into a polypeptide. Expression produces a “gene product.” As used herein, the gene product may be a nucleic acid, such as RNA produced by the transcription of a gene, e.g. The gene product may be a nucleic acid or polypeptide translated from a transcript. The gene product described herein further comprises a nucleic acid by post-transcriptional modification, e.g., polyadenylation or splicing, or a polypeptide by post-translational modification, e.g., phosphorylation, methylation, glycosylation, lipid addition, association with other protein subunits, or proteolytic cleavage.

[0039] As used herein, the term "identity" refers to the preservation of the entire monomer between polymer molecules, for example, polynucleotide molecules. The term "identical" without any additional modifiers, for example, that polynucleotide A is identical to polynucleotide B, means that the polynucleotide sequences are 100% identical (100% sequence identity). Describing two sequences as, for example, "70% identical" is equivalent to describing them as having, for example, "70% sequence identity."

[0040] For example, the calculation of identity (percentage) between two polypeptide or polynucleotide sequences can be performed by aligning the two sequences for optimal comparison purposes (for example, for optimal alignment, a gap may be introduced in one or both of the first and second polypeptide or polynucleotide sequences, and sequences that are not identical for comparison purposes may be ignored). In a specific embodiment, the length of the sequence aligned for comparison purposes is at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or 100% of the length of the reference sequence. Subsequently, the amino acids at corresponding amino acid positions, or in the case of polynucleotides, the bases, are compared.

[0041] If a specific position in the first sequence is occupied by the same amino acid or nucleotide as the corresponding position in the second sequence, the molecules are identical at that position. The identity (percentage) between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps that need to be introduced for optimal alignment of the two sequences and the length of each of the gaps. Sequence comparison and the determination of the identity (percentage) between the two sequences can be performed using a mathematical algorithm.

[0042] Suitable software programs that can be used to align 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 U.S. government’s National Center for Biotechnology Information BLAST website (blast.ncbi.nlm.nih.gov). bl2seq performs a comparison between two sequences using the BLASTN or BLASTP algorithms. BLASTN is used to compare nucleic acid sequences, whereas BLASTP is used to compare amino acid sequences. Other suitable programs include, for example, Needle, Stretcher, Water, or Matcher, which are part of the bioinformatics program EMBOSS suite and are also available from the European Bioinformatics Institute (EBI) at www.ebi.ac.uk / Tools / psa.

[0043] Sequence alignment can be performed using methods known in the art, such as MAFFT, Clustal (ClustalW, Clustal X or Clustal Omega), MUSCLE, etc.

[0044] Different regions within a single polynucleotide or polypeptide target sequence aligned with the polynucleotide or polypeptide reference sequence may each have their own sequence identity (percentage). Note that sequence identity (percentage) values ​​are rounded up or down to the nearest first decimal place. For example, 80.11, 80.12, 80.13, and 80.14 are rounded down to 80.1, and 80.15, 80.16, 80.17, 80.18, and 80.19 are rounded up to 80.2. Also, note that length values ​​will always be integers.

[0045] In a specific embodiment, the identity (percentage, %ID) of a first amino acid sequence (or nucleic acid sequence) with respect to a second amino acid sequence (or nucleic acid sequence) is calculated as %ID = 100 Х (Y / Z), where Y is the number of amino acid residues (or nucleobases) scored as identical matches in the alignment of the first and second sequences (when aligned by visual inspection or by a specific sequence alignment program) and Z is the total number of residues in the second sequence. If the length of the first sequence is longer than that of the second sequence, the identity (percentage) of the first sequence with respect to the second sequence will be higher than the identity (percentage) of the second sequence with respect to the first sequence.

[0046] Those skilled in the art will understand that the generation of sequence alignments to calculate sequence identity (percentage) is not limited to binary sequence-sequence comparisons processed entirely by primary sequence data. It will also be understood that sequence alignments can be achieved by integrating sequence data with data from heterogeneous sources, such as structural data (e.g., crystallographic protein structure), functional data (e.g., mutation locations), or phylogenetic data. A suitable program for achieving multiple sequence alignments by integrating heterogeneous data is T-Coffee, available at www.tcoffee.org and, alternatively, from, for example, EBI. It will also be understood that the final alignment used to calculate sequence identity (percentage) can be systematized automatically or manually.

[0047] As used herein, the term "intron" refers to a portion of DNA within a gene (intermediate sequence) that is spliced ​​from mRNA transcribed from the gene before being released from the cell nucleus, and which does not code for a portion of the protein produced by the gene. "Intron sequence" refers to the nucleic acid sequence of an intron. Such sequences are also referred to herein as "non-coding nucleic acid sequences." Accordingly, introns are regions of DNA sequences that are transcribed along with the coding sequence (exon) but are removed during the formation of mature mRNA.

[0048] As used herein, the term “intron fragment” refers to a fragment derived from a full-length intron sequence (e.g., a full-length EF-1α intron A sequence). The term “fragment” implies the exclusion of the full-length intron. In some embodiments, the “intron fragment” comprises the minimum number of nucleotides or constructs necessary to achieve an expression level exceeding that achieved by a corresponding construct in which all nucleotides of EF-1α intron A are missing. Accordingly, the intron fragment of this disclosure (also referred to herein as “non-translating nucleic acid sequence”) is not particularly limited as long as it comprises a fragment of the EF-1α intron and can increase the expression of the foreign gene. As demonstrated herein, in some embodiments, the intron fragment (i.e., non-translating nucleic acid sequence) can increase the expression of the foreign gene by increasing the transcription of the foreign gene. Accordingly, in some embodiments, the intron fragment described herein may be a non-translating regulatory element.

[0049] The terms “isolated,” “purified,” “extracted,” and their grammatical variations used herein are used interchangeably and refer to the state of a desired composition of the present disclosure, e.g., a TAFA protein, or a preparation of a polypeptide comprising a fragment or variant thereof, or a polynucleotide comprising a nucleic acid sequence encoding thereof, that has undergone one or more purification processes. In some embodiments, isolation or purification as used herein is a process of removing or partially removing (e.g., fractionating) a composition of the present disclosure, e.g., a polypeptide or polynucleotide described herein, from a sample containing contaminants.

[0050] In some embodiments, the isolated composition has no detectable undesirable activity, or otherwise, the level or amount of undesirable activity is below an acceptable level or amount. In other embodiments, the isolated composition has an amount and / or concentration of the desired composition of the present disclosure above an acceptable amount and / or concentration and / or activity. In other embodiments, the isolated composition is concentrated relative to the starting material from which the composition is obtained. This concentration 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% compared to the starting material.

[0051] In some embodiments, the isolated preparation is substantially free of residual biological products. In some embodiments, the isolated preparation is free of any biological contaminants in 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%. Residual biological products may include abiotic substances (including chemicals) or unwanted nucleic acids, proteins, lipids, or metabolites.

[0052] As used herein, the term "linked" refers to a first amino acid sequence or polynucleotide sequence that is covalently or non-covalently joined to a second amino acid sequence or polynucleotide sequence, respectively. The first amino acid or polynucleotide sequence may be directly joined or juxtaposed to the second amino acid or polynucleotide sequence, or alternatively, an intervening sequence may covalently join the first sequence to the second sequence. For example, since the TAFA protein is a polypeptide comprising a form in which fragments 1, 2, and 3 are joined, the fragments of the TAFA protein may be a form in which fragment 2 and fragment 3 are joined or fragment 5 and fragment 7 are joined. The term "linked" means not only the fusion of the first polynucleotide sequence to the second polynucleotide sequence at the 5'-terminus or 3'-terminus, but also includes inserting the entire first polynucleotide sequence (or the second polynucleotide sequence) between any two nucleotides within the second polynucleotide sequence (or the first polynucleotide sequence). The first polynucleotide sequence may be linked to the second polynucleotide sequence by a phosphodiester bond or a linker. The linker may be, for example, a polynucleotide.

[0053] "Nucleic acid," "nucleic acid molecule," "nucleotide sequence," "polynucleotide," and their grammatical variations are used interchangeably and refer to a sequence of nucleotides linked by phosphodiester bonds. Polynucleotides are indicated in this specification from 5' to 3'. Polynucleotides in this disclosure may be deoxyribonucleic acid (DNA) molecules or ribonucleic acid (RNA) molecules. Nucleotide bases are indicated in this specification by single-character codes such as adenine (A), guanine (G), thymine (T), cytosine (C), inosine (I), and uracil (U).

[0054] As used herein, the terms “operably linked” or “operably linked” mean that the DNA sequences to be linked are located adjacent to perform a desired function. For example, if a specific promoter helps initiate the transcription of a coding sequence (e.g., a foreign gene), such a promoter may be operably linked to the coding region. As long as this functional relationship is maintained, the promoter and the coding region do not necessarily have to be located adjacent to each other.

[0055] The terms “pharmaceuticalally acceptable carrier,” “pharmaceutically acceptable excipient,” and their grammatical variations include all preparations approved by U.S. federal regulatory agencies or listed in the United States Pharmacopoeia for use in animals, including humans, as well as all carriers or diluents that do not cause the occurrence of undesirable physiological effects to the extent that they would prevent administration of the composition to a subject, and do not impair the biological activity and properties of the administered complex. It includes excipients and carriers that are useful for manufacturing pharmaceutical compositions, are generally safe, non-toxic, and desirable.

[0056] As used herein, the term “pharmaceutical composition” refers to one or more of the compositions described herein (e.g., polypeptides, polynucleotides, vectors, cells, and / or recombinant viruses) that are mixed or blended with or suspended in one or more other chemical components, such as pharmaceutically acceptable carriers and excipients.

[0057] As used herein, the terms “promoter” and “promoter sequence” refer to compatible DNA sequences capable of regulating the expression of a coding sequence or functional RNA. Generally, the coding sequence is located at the 3’ position relative to the promoter sequence. A promoter may be entirely derived from a natural gene, may consist of different elements derived from different promoters found in nature, or may further include a synthetic DNA portion. Those skilled in the art understand that different promoters may direct gene expression in different tissues or cell types, at different developmental stages, or in response to different environmental or physiological conditions. A promoter that causes a gene to be mostly expressed in most host cell types is commonly referred to as a “constitutive promoter.” A promoter that causes a gene to be expressed in a specific cell type is commonly referred to as a “cell-specific promoter” or a “tissue-specific promoter.” Promoters that cause gene expression during specific developmental or cell differentiation stages are commonly referred to as "development-specific promoters" or "cell differentiation-specific promoters." Promoters that induce gene expression after exposing or treating cells with promoter-inducing agents, biological molecules, chemicals, ligands, light, etc., are commonly referred to as "inducible promoters" or "regulatory promoters." Furthermore, since the precise boundaries of regulatory sequences were not fully defined in most cases, it is recognized that DNA fragments of different lengths can possess the same promoter activity.

[0058] The promoter sequence is typically bounded by a transcription initiation site at its 3' end and extends upstream (in the 5' direction) to include the minimum number of bases or elements required to initiate transcription at a detectable level above the background. Within the promoter sequence, a protein binding domain (common sequence) responsible for binding RNA polymerase will be found, as well as a transcription initiation site (conveniently defined, for example, by mapping to nuclease S1). In some embodiments, the promoters available in this disclosure include tissue-specific promoters.

[0059] As used herein, the terms “gene regulatory region” or “regulatory region” refer to a nucleotide sequence located upstream (5’ non-coding sequence), within, or downstream (3’ non-coding sequence) of a coding region that affects the transcription, RNA processing, stability, or translation of the relevant coding region. The regulatory region may include a promoter, a translation leader sequence, an intron, a polyadenylation recognition sequence, an RNA processing site, an effector binding site, or a stem-loop structure. When the coding region is intended for expression in eukaryotic cells, the polyadenylation signal and transcription termination sequence will typically be located at the 3’ relative to the coding sequence.

[0060] In some embodiments, the polynucleotides described herein (e.g., including foreign genes and non-translating nucleic acid sequences encoding polypeptides comprising amino acid sequences of TAFA proteins, fragments thereof, or variants thereof) may include other expression (e.g., transcription) regulatory elements operably associated with a promoter and / or one or more coding regions. In the operable association, the coding region for a gene product is associated with one or more regulatory regions in such a manner that the expression of said gene product is placed under the influence or control of the regulatory region(s). For example, the coding region and the promoter are "operably associated" if the induction of the promoter function causes the transcription of mRNA encoding the gene product encoded by said coding region, and the linkage characteristics between said promoter and the coding region do not interfere with the promoter's ability to direct the expression of the gene product or with the ability of the DNA template to be transcribed. Other expression regulatory elements other than the promoter, e.g., enhancers, operators, repressors, and transcription termination signals, may also be operably associated with the coding region directing the expression of the gene product.

[0061] As used herein, the terms “subject,” “patient,” “individual,” and “host,” and variants thereof, refer to any mammalian subject to which any of the compositions described herein (e.g., polypeptides, polynucleotides, recombinant expression constructs, vectors, cells, pharmaceutical compositions, or recombinant viruses) are administered. Non-limiting examples include mammals other than humans that require diagnosis, treatment, or therapy. Other non-limiting examples include humans, livestock (e.g., dogs, cats, etc.), farm animals (e.g., cattle, sheep, pigs, horses, etc.), and laboratory animals (e.g., monkeys, rats, mice, rabbits, guinea pigs, etc.), and particularly humans that require diagnosis, treatment, or therapy. The methods described herein are applicable to both human prophylaxis or treatment and veterinary use.

[0062] The phrase "objects requiring" as used in this specification includes subjects such as mammalian subjects who would benefit from the administration of the compositions described in this specification.

[0063] As used herein, the term “therapeutic effective dose” is an amount of a reagent or pharmaceutical complex comprising a composition of the present disclosure (e.g., a TAFA protein, or a polypeptide comprising a fragment or variant thereof, or a polynucleotide encoding thereof) sufficient to obtain a desired therapeutic effect, pharmacological and / or physiological effect on a target requiring treatment. The therapeutic effective dose may be a “preventive effective dose” because prevention can be regarded as treatment.

[0064] As used herein, the term “foreign gene” refers to at least one polynucleotide encoded in a recombinant expression construct (e.g., a polynucleotide encoding a polypeptide comprising a TAFA protein, or a fragment thereof or a variant thereof, or a polynucleotide encoding a fusion polypeptide (e.g., a polypeptide in which another polypeptide (e.g., Fc, albumin, or other types of peptides, etc.) is fused to a TAFA protein or a fragment thereof) or a polynucleotide region or an expression product of said polynucleotide or polynucleotide region, a polypeptide or a polypeptide encoding a polynucleotide or a multi-polypeptide, or a promoting or regulatory nucleic acid. In some embodiments, said foreign gene may be heterogeneous to the cell into which it is inserted (or transduced) (i.e., not naturally expressed in the cell).

[0065] As used herein, the terms “to treat,” “treatment,” or “treating” refer to, for example, a reduction in the severity of a disease or condition, a reduction in the duration of the disease, the improvement or elimination of one or more symptoms associated with the disease or condition, or providing beneficial effects to a subject having a disease or condition without necessarily curing the disease or condition. The terms also include preventing or preventing a disease or condition or its symptoms.

[0066] As used herein, the terms “vector” or “construct” refer to any vehicle into which a nucleic acid or gene may be inserted, such as a delivery vehicle into which a nucleic acid sequence may be inserted and replicated. The nucleic acid sequence into which the nucleic acid sequence may be inserted may be exogenous or heterogeneous. The nucleic acid sequence may be a foreign gene. Examples of constructs include, but are not limited to, plasmids, cosmids, and viruses (e.g., AAV). Those skilled in the art may construct said vector or construct by standard recombinant techniques (Maniatis, et al., *Molecular Cloning*, *A Laboratory Manual*, Cold Spring Harbor Press, Cold Spring Harbor, NY, 1988; and Ausubel et al., *Current Protocols in Molecular Biology*, John, Wiley & Sons, Inc, NY, 1994, etc.). As used herein, the terms “expression vector” or “expression construct” mean a vector or construct comprising a nucleotide sequence encoding at least a portion of the gene product being transcribed. In some cases, the RNA molecule is subsequently translated into a protein, polypeptide, or peptide. The expression construct may include various regulatory elements. Along with regulatory sequences that regulate transcription and translation, the vector and expression vector may also include nucleotide sequences that provide other functions. Viruses available in the present invention include, but are not limited to, retroviruses, herpes simplex virus, lentivirus, poxvirus, vaccinia virus, rhabdovirus, adenovirus, helper-dependent adenovirus, adeno-associated virus (AAV), etc.

[0067] The vector may be engineered to code for a selective marker or reporter provided for the selection or identification of cells incorporating the vector. The expression of the selective marker or reporter enables the identification and / or selection of host cells that incorporate and express other coding regions included in the vector. Examples of selective marker genes known and used in the art include genes that confer resistance to ampicillin, streptomycin, gentamicin, kanamycin, hygromycin, vialaphos herbicides, sulfonamides, etc.; and genes used as phenotypic markers, namely anthocyanin regulatory genes, isofentanyl transferase genes, etc. Examples of reporters known and used in the art include luciferase (Luc), green fluorescent protein (GFP), chloramphenicol acetyltransferase (CAT), β-galactosidase (LacZ), β-glucuronidase (Gus), etc. Selective markers may also be considered as reporters.

[0068] Generally, recombinant adeno-associated virus (AAV) is produced by triple transfection of a host cell (e.g., HEK293 cell). It consists of 1) an AAV construct plasmid with a gene expression cassette alongside inverted terminal repeats (ITRs), 2) a "Rep-Cap plasmid" providing the Rep protein required for the replication of the adeno-associated virus genome and the capsid protein that constitutes the viral particle, and finally 3) a "helper plasmid" providing the adenovirus proteins (E2a, E4) and RNA (VA RNA) that help the AAV life cycle. AAV is produced when these three types of plasmids are transfected into cells that provide the adenovirus E1 gene (e.g., HEK293 cells or cells derived from HEK293 cells (293T, 293FT, 293F / FreeStyle 293-F, 293E, Expi293 / Expi293F, HEK293S, HEK293A / 293H, 293T / 17, etc.)).

[0069] In this specification, the term “double helper plasmid” means a plasmid capable of providing two or more of the requirements for generating AAV in a cell. As is apparent from the present disclosure, in some aspects, the double helper plasmid described herein provides the requirements described above: 2) “Rep-Cap plasmid” and 3) “helper plasmid.” For example, in some aspects, the double helper plasmid comprises a rep gene, a cap gene, an E2a gene, an E4 gene, and a VA RNA gene.

[0070] That is, AAV is produced when 1) an AAV construct plasmid with a gene expression cassette next to ITRs, and 2) a double helper plasmid are transfected into a cell providing the E1 gene of an adenovirus.

[0071] The double helper plasmid described herein not only contains the gene described above, but said gene may also be arranged in a specific configuration within the plasmid. For example, in some aspects, the E2a gene, the E4 gene, and the VA RNA gene are sequentially linked within the double helper plasmid, and the rep gene and the cap gene (collectively referred to herein as the “rep-cap gene”) are sequentially linked in a clockwise direction (from 5’ to 3’) between the 5’ end of the E2a gene and the 3’ end of the VA RNA gene. More specifically, in some aspects, the 5’-end of the rep-cap gene is linked to the 5’-end of the E2a gene, where the 3’-end of the rep-cap gene is linked to the 3’ end of the VA RNA gene. In some aspects, the E2a gene, the E4 gene, and the VA RNA gene are linked sequentially, and the rep-cap gene is linked counterclockwise (from 3' to 5') between the 5'-end of the E2a gene and the 3'-end of the VA RNA gene. More specifically, in some aspects, the 3'-end of the rep-cap gene is linked to the 5'-end of the E2a gene, where the 5'-end of the rep-cap gene is linked to the 3'-end of the VA RNA gene.

[0072] In this specification, the term "cell" includes eukaryotic and prokaryotic cells and refers to any transgenic cell capable of replicating the vector or expressing the gene encoded by the vector. The cell may be transfected, transduced, or transformed by the vector, which refers to the process in which an exogenous polynucleotide (nucleic acid molecule) is delivered or introduced into a host cell. In this specification, the term "transformation" is used to include the meanings of transfected and transduced, or may be used interchangeably.

[0073] The (host) cells of the present invention are not limited, but preferably insect cells or mammalian cells, more preferably Sf9 in the case of insect cells, and HEK293 cells, HeLa cells, ARPE-19 cells, RPE-1 cells, HepG2 cells, Hep3B cells, Huh-7 cells, C8D1a cells, Neuro2A cells, CHO cells, MES13 cells, BHK-21 cells, COS7 cells, COP5 cells, A549 cells, MCF-7 cells, HC70 cells, HCC1428 cells, BT-549 cells, PC3 cells, LNCaP cells, Capan-1 cells, Panc-1 cells, MIA PaCa-2 cells, SW480 cells, HCT166 cells, LoVo cells, A172 cells, MKN-45 cells, MKN-74 cells, Kato-III cells, NCI-N87 cells, HT-144 cells, SK-MEL-2 cells, SH-SY5Y cells, C6 cells, HT-22 cells, PC-12 cells, NIH3T3 cells, etc. may be used. In some embodiments, the host cell is a recombinant host cell. In some embodiments, the host cell is an isolated host cell.

[0074] As used herein, the terms “family having sequence similarity 19,” “FAM19,” or “TAFA” refer to proteins belonging to the TAFA family (also known as the FAM19 family) of five proteins that are primarily expressed in the brain and spinal cord, and are also referred to as TAFA proteins or TAFA polypeptides. FAM19A1 is also known as TAFA1, FAM19A2 as TAFA2, FAM19A3 as TAFA3, FAM19A4 as TAFA4, and FAM19A5 as TAFA5.

[0075] The human TAFA1 gene encodes a sequence of 133 amino acids. Although there is disagreement regarding the lengths of the signal peptide and the mature protein, the human TAFA1 protein is expected to consist of a 35-amino acid signal peptide and a 98-amino acid mature protein. TAFA1 shows high expression in the frontal, temporal, occipital, and parietal cortices, and low expression in the basal ganglia, thalamus, and cerebellum. Cell experiments have revealed that TAFA1 influences the determination of differentiation fate in neural stem cells, inhibiting the differentiation of neural stem cells into astrocytes while promoting their differentiation into neurons. Through TAFA1 Knock-Out (KO) mouse experiments, it has been shown that TAFA1 can regulate motor activity, anxiety-related behavior, learning and memory, and somatosensory functions.

[0076] The human TAFA2 gene encodes a sequence of 131 amino acids. Although there is disagreement regarding the lengths 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 abundantly expressed in the larynx, frontal cortex, and medulla oblongata within the central nervous system. It is known that injecting recombinant TAFA2 protein into the third ventricle of mice increases food intake and meal frequency, as well as energy expenditure and the respiratory exchange rate. Therefore, this suggests that TAFA2 may play a role in regulating food intake and energy metabolism. Furthermore, it is known that inhibiting TAFA2 in zebrafish and mice increases anxiety-related behaviors.

[0077] The human TAFA3 gene encodes a sequence of 133 amino acids. Although there is disagreement regarding the lengths of the signal peptide and the mature protein, the human TAFA3 protein is expected to consist of a signal sequence of 30 amino acids and a mature protein of 103 amino acids. It is known that TAFA3 expression increases in microglia in a mouse model of transient focal ischemia, and that microglia treated with TAFA3 are polarized into anti-inflammatory microglia. Furthermore, when TAFA3 was KO'd in a mouse model, three major behavioral deficits observed in the autism spectrum—such as reduced response to social novelty, impaired social communication, and increased repetitive behaviors—appeared, suggesting that TAFA3 is involved in the normal functioning of social relationship formation.

[0078] The human TAFA4 gene encodes a sequence of 140 amino acids, and while there is disagreement regarding the lengths of the signal peptide and 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 (C-low-threshold mechanoreceptors) and appears to reduce pain by regulating the activity of interneurons.

[0079] The human TAFA5 gene encodes a sequence of 132 amino acids. 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 signal sequence of 43 amino acids and a mature protein of 89 amino acids. TAFA5 shows high expression in the basal ganglia and cerebellum. It is known that TAFA5 expression increases in the hypothalamus of mice in response to inflammatory stimuli such as TNF-α, and that when TAFA5 is KO'd, the decrease in food intake, body weight loss, and increase in inflammatory cytokines caused by TNF-α are partially restored.

[0080] In the case of the above TAFA1 to TAFA4 mature proteins, they exhibit high sequence identity (e.g., human TAFA4 has 73.7% sequence identity with TAFA1, 85.3% sequence identity with TAFA2, and 81.1% sequence identity with TAFA3). Meanwhile, TAFA5 exhibits low sequence identity with TAFA1 to 4 (e.g., human TAFA5 has 48.9% sequence identity with TAFA1, 51.1% sequence identity with TAFA2, 47.7% sequence identity with TAFA3, and 50.0% sequence identity with TAFA4).

[0081] Accordingly, in this specification, the terms "TAFA protein" or "TAFA polypeptide" are used to refer to TAFA1 to TAFA4 proteins or polypeptides, excluding TAFA5.

[0082] The human TAFA4 (TAFA Chemokine Like Family Member 4) protein (Sequence No. 299) is one of the TAFA protein family (TAFA1-5) that is abundantly expressed throughout brain regions. The above TAFA4, composed of 140 amino acids, has 95 or 93 amino acids that are evolutionarily very well conserved in vertebrates, excluding 45 or 47 amino acids that contain the signal sequence in the preceding portion. Furthermore, among the TAFA protein family, it has been confirmed that the interspecies sequence identity of TAFA1 to 4 is very well conserved in vertebrates. i) The mature human TAFA4 amino acid sequence exhibits more than 90% sequence identity compared to mammalian and amphibian TAFA4, more than 95% sequence identity compared to avian TAFA4, and more than 85% sequence identity compared to reptile and fish TAFA4; ii) The human TAFA1 amino acid sequence exhibits more than 90% sequence identity compared to mammalian and amphibian TAFA1, more than 95% sequence identity compared to avian TAFA1, and more than 85% sequence identity compared to reptile and fish TAFA1; iii) The human TAFA2 amino acid sequence exhibits more than 90% sequence identity compared to mammalian and amphibian TAFA2, more than 95% sequence identity compared to avian TAFA2, and more than 80% sequence identity compared to reptile and fish TAFA2; iv) The human TAFA3 amino acid sequence exhibits more than 75% sequence identity compared to mammalian and amphibian TAFA3, more than 80% sequence identity compared to avian TAFA3; It shows more than 75% sequence identity compared to TAFA3 in reptiles and fish, confirming that it exhibits very high sequence identity regardless of species.

[0083] As used in this specification, the term "inner ear disease" refers to any disease, disorder, or condition associated with damage or abnormality of the auditory system or the balance system (vestibular system) in the inner ear. The inner ear disease includes two types of organs in which ribbon synapses exist within the inner ear: i) auditory system disease or dysfunction in the cochlea (inner hair cell (IHC) and outer hair cell (OHC)) and ii) balance system disease or dysfunction (vestibular synaptopathy) in the vestibular organs (oval capsule, saccule, and semicircular canals).

[0084] The above auditory disorders or dysfunctions include, but are not limited to, hidden hearing loss, noise-induced synaptopathy, age-related synaptopathy, tinnitus, sensorineural hearing loss (SNHL), or combinations thereof.

[0085] The above balance system (vestibular system) disorders or dysfunctions include, but are not limited to, chronic vestibular decline, transient vestibular syncope, acute vestibular neuropathy, age-related vestibular decline, autoimmune inner ear disease, auditory neuropathy, or combinations thereof.

[0086] In the present invention, the term "sensorineural hearing loss" refers to hearing loss caused by a disorder in the sound detection function of the cochlea, or by a dysfunction of the auditory nerve or the central nervous system that transmits auditory stimuli to the brain. That is, it may refer to hearing loss caused by partial or total damage or abnormality to auditory receptors (inner hair cells and outer hair cells), auditory nerve fibers, spiral ganglion cells, ribbon synapses, or the central auditory pathway. Preferably, the sensorineural hearing loss may refer to hearing loss excluding conductive hearing loss (for example, hearing loss that occurs when there is a disorder in the process of sound being transmitted from the outer ear and middle ear to the inner ear, even though the functions of the cochlea and auditory nerve are normal). In some aspects, the sensorineural hearing loss includes non-hereditary hearing loss. Examples of non-hereditary hearing loss include, but are not limited to, ototoxic hearing loss, noise-induced hearing loss (NIHL), hearing loss due to infection (e.g., congenital CMV, congenital rubella, syphilis, bacterial meningitis, labyrinthitis, etc.), hearing loss due to immune abnormalities or inflammation (autoimmune inner ear disease (AIED), sarcoidosis, etc.), hearing loss due to trauma (head trauma, skull fracture, etc.), presbycusis, sudden sensorineural hearing loss (SSNHL), endolymphatic hydrops, or combinations thereof. In some aspects, the sensorineural hearing loss includes hereditary hearing loss. Hereditary hearing loss includes nonsyndromic hearing loss (Nonsyndromic HHL) or syndromic hearing loss (Syndromic HHL).Non-syndromic hearing loss can be caused by problems with hundreds of genes or combinations thereof, such as GJB2(Cx26), GJB6(Cx30), SLC26A4, OTOF, TMC1, MYO7A, POU3F4, MTRNR1, and ACTG1. Examples of syndromic hearing loss include, but are not limited to, Auditory Neuropathy Spectrum Disorder, Usher Syndrome, Pendred Syndrome, Waardenburg syndrome, Alport syndrome, Branchio-oto-renal (BOR) syndrome, CHARGE syndrome, Jervell and Lange-Nielsen syndrome, Stickler syndrome, Treacher Collins syndrome, mitochondrial syndromes (e.g., MERRF, MELAS, etc.)) or combinations thereof. In some aspects, inner ear disorders are associated with partial hearing loss. For example, compared to a reference subject (e.g., a corresponding subject not suffering from an inner ear disease), the hearing ability of a person suffering from an inner ear disease (e.g., described herein) is reduced by at least about 5%, 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%, or about 100%. In some aspects, inner ear disease is associated with complete hearing loss. The subject's hearing may be evaluated using any suitable method known in the art.Non-limiting examples of methods that can be used to assess whether a subject has an inner ear disorder include auditory brainstem response (ABR), modulated otoacoustic emission (DPOAE), pure tone audiometry (PTA), extended tone audiometry (ETA), speech intelligibility (SDS), word recognition under noise (WIN), tinnitus function index (TFI), and tinnitus intensity (TL).

[0087] As used herein, the term “tinnitus” refers to the perception of hallucinatory hearing in the absence of external auditory stimulation. The two main types of tinnitus include (1) “objective tinnitus,” which is caused by sounds originating from somewhere in the body (e.g., turbulence in the head or muscle contractions); and (2) “subjective tinnitus,” which is the perception of meaningless sounds in the absence of physical sounds. Unlike subjective tinnitus, which is heard only by the person with the tinnitus, objective tinnitus is rare and may be heard by an observer. Tinnitus sounds may appear in various forms, such as buzzing, ringing, or other various sounds. Tinnitus may occur continuously or sporadically in one or both ears. As is evident from the present invention, in some aspects, inner ear conditions that can be treated by the present invention (e.g., sensorineural hearing loss) are associated with tinnitus. In some aspects, inner ear conditions that can be treated by the present invention (e.g., sensorineural hearing loss) are not associated with tinnitus.

[0088] Signal transmission between auditory cells and spiral ganglion neurons is mediated by a unique structure called the ribbon synapse in the inner ear. Ribbon synapses are a key structure that enables the rapid and continuous transmission of auditory signals and are essential for the normal perception of sound. However, ribbon synapses can be damaged by various factors such as noise, aging, and drugs, and it has been reported through several studies that such damage is closely associated with hearing loss (Paul A Fuchs et al., "The afferent synapse of cochlear hair cells", Curr Opin Neurobiol., 13(4):452-458, (Aug. 2003), Jukic A et al., "Kujawa SG et al., “Adding insult to injury: cochlear nerve degeneration after "temporary" noise-induced hearing loss”, J Neurosci., 11;29(45):14077-85, (Nov. 2009), Fernandez KA et al., "Trk agonist drugs rescue noise-induced hidden hearing loss" JCI Insight, 8;6(3):e142572, (Feb. 2021)).

[0089] As used herein, the term “ribbon synapse” refers to a type of neural synapse characterized by the presence of an electron-integrated structure (i.e., “synaptic ribbon”) containing numerous synaptic vesicles (containing various neurotransmitters) located near the active region (i.e., the neurotransmitter release site). Ribbon synapses are characterized by tight vesicle-calcium channel couplings that facilitate rapid neurotransmitter release and sustained signal transmission. Ribbon synapses undergo cycles of exocytosis and endocytosis in response to stepwise changes in membrane potential. Unless otherwise specified, ribbon synapses described herein serve in relation to the auditory system and / or the sedative system (equilibrium system) within the inner ear. In the auditory system, ribbon synapses are located at i) the site where inner hair cells (IHCs) bind with spiral ganglion neurons and ii) the site where outer hair cells (OHCs) bind with Type II spiral ganglion neurons (Coate, Thomas M., M. Katie Scott, and Mansa Gurjar.Synapse 73.5 (2019): e22087.).

[0090] As used herein, the term "auditory system" means a sensory system for hearing of a subject (e.g., a human subject).

[0091] The term "inner hair cell" (IHC) refers to a type of cochlear hair cell (i.e., sensory cells of the auditory system). At least in humans, cochlear hair cells consist of one row of inner hair cells and three rows of outer hair cells. Inner hair cells are actual sensory receptors (converting sound vibrations in the cochlear fluid into electrical signals), and 95% of the auditory nerve fibers heading to the brain originate from this subgroup.

[0092] The term "neuron" includes electrically excitable cells that process and transmit information through electrical and chemical signals. A typical neuron consists of a cell body (soma), dendrites, and an axon (also referred to herein as a "nerve fiber"). The soma (cell body) of a neuron contains the nucleus. The dendrites of a neuron are extensions of the cell with many branches, where most of the input to the neuron originates. The axon is a finer, cable-like projection extending from the soma, which transmits nerve signals from the soma and conveys specific types of information back to the soma.

[0093] As used herein, the term "nerve fiber" refers to a part of a neuron, namely the axon, which carries an action potential from one end of the neuron to the other. In some aspects, nerve fibers include "cochlear nerve fibers." Cochlear nerve fibers originate from neurons in the spiral ganglion and extend peripherally to the cochlear hair cells and centrally to the cochlear nucleus of the brainstem. Cochlear nerve fibers mediate hearing.

[0094] As used in this specification, the term "spiral ganglion" refers to a sensory ganglion of the cochlear nerve. The spiral ganglion is the "primary neuron of the auditory nerve" that converts mechanical sound stimuli from hair cells into electrical nerve signals and transmits them to the brain, serving as the starting point of the auditory pathway. The cells of the spiral ganglion send fibers peripherally to the cochlear hair cells and centrally to the cochlear nucleus of the brainstem. Spiral ganglia are classified into Type I, which is connected to inner hair cells (IHC), and Type II, which is connected to outer hair cells (OHC). Representative diseases resulting from damage to the spiral ganglion itself, neurodegeneration, or branching disorders include, but are not limited to, Auditory Neuropathy Spectrum Disorder (ANSD), Noise-induced hearing loss, Age-related hearing loss (ARHL), diabetic auditory neuropathy, Spiral ganglionitis, vestibular schwannoma, or combinations thereof.

[0095] As used herein, the term "cochlea" refers to a spiral cavity in the inner ear that resembles a snail shell and contains nerve endings essential for hearing. The cochlea contains three fluid-filled chambers: the tympanic system, the vestibular system (containing perilymph), and the mesosystem (containing endolymph). The tympanic system and the vestibular system are adjacent to each other and merge at the cochlear opening, which is the end of the snail shell. The stapes transmits vibrations to the oval window (an elliptical window) located on the outside of the cochlea, causing the perilymph of the vestibular system to vibrate. The perilymph vibrates the endolymph of the mesosystem, inducing movement of the hair bundles of hair cells, which are acoustic sensor cells that convert vibrations into electrical potentials. The hair cells are arranged in four rows within the organ of Corti along the entire length of the cochlear coil. Three rows consist of outer hair cells (OHCs), and one row consists of inner hair cells (IHCs). The IHCs provide the primary neural output of the cochlea. On the other hand, outer hair cells primarily receive neural input from the brain, and this influences motility as part of the cochlea's mechanical preamplifier.

[0096] Meanwhile, the key structural protein of inner ear ribbon synapses is RIBEYE, which consists of two domains: the A-domain (inherent domain) and the B-domain = CtBP2. In other words, CtBP2 is a key factor in maintaining the structural and functional integrity of ribbon synapses, contributing to rapid and efficient signal transmission by binding vesicles to the synaptic ribbon and preparing for exocytosis. Furthermore, as a marker protein specific to ribbon synapses, the degree of ribbon synaptic damage can be histologically assessed by counting it; counting CtBP2 is almost essential in the study of auditory synaptopathy. (Manickam V et al., “Macrophages Promote Repair of Inner Hair Cell Ribbon Synapses following Noise-Induced Cochlear Synaptopathy”, J Neurosci., 22;43(12):2075-2089 (03,2023))

[0097] As demonstrated in the present invention, in some aspects, a polypeptide comprising the amino acid sequence of a TAFA protein or a fragment thereof or a variant thereof described herein may increase the neurite growth of a neuron (e.g., a spiral ganglion neuron). As used herein, the term “neurite growth” refers to the process in which new projections (axons, dendrites, or both) are generated as a neuron grows. Accordingly, some aspects of the present invention relate to a method for increasing the neurite growth of a spiral ganglion neuron of a subject in need thereof, comprising administering the polypeptide, nucleic acid molecule, vector, or recombinant virus particle described herein to the subject. In some aspects, neurite growth increases by 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%, or at least about 100% after administration of the polypeptide, nucleic acid molecule, vector, or recombinant virus particle. In some aspects, neurite growth increases by at least about 1.5 times, at least about 2 times, at least about 3 times, at least about 4 times, at least about 5 times, at least about 6 times, at least about 7 times, at least about 8 times, at least about 9 times, at least about 10 times, at least about 12.5 times, at least about 15 times, at least about 20 times, at least about 25 times, at least about 30 times, at least about 35 times, at least about 40 times, at least about 45 times, or at least about 50 times after administration of the polypeptide, nucleic acid molecule, vector, or recombinant virus particle. In some aspects, the polypeptide, nucleic acid molecule, vector, or recombinant virus particle described herein may reverse the inhibition of neurite growth.

[0098] In some aspects, the polypeptide, nucleic acid molecule, vector, or recombinant virus particle described herein may promote synapse formation. As used herein, the term “synapse formation” refers to the process of synapse formation between neurons (e.g., spiral ganglion neurons). Accordingly, in some aspects, the present invention provides a method for increasing synapse formation in a subject in need thereof, comprising administering to a subject a polypeptide comprising the amino acid sequence of a TAFA protein or a fragment thereof or a variant thereof described herein, a nucleic acid molecule encoding said polypeptide, a vector comprising said nucleic acid molecule, or a recombinant virus particle comprising said vector and a capsid protein. In some aspects, synapse generation increases by 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%, or at least about 100% after administration of the polypeptide, nucleic acid molecule, vector, or recombinant virus particle. In some aspects, synapse generation after administration of the polypeptide, nucleic acid molecule, vector, or recombinant virus particle is increased by at least about 1.5 times, at least about 2 times, at least about 3 times, at least about 4 times, at least about 5 times, at least about 6 times, at least about 7 times, at least about 8 times, at least about 9 times, at least about 10 times, at least about 12.5 times, at least about 15 times, at least about 20 times, at least about 25 times, at least about 30 times, at least about 35 times, at least about 40 times, at least about 45 times, or at least about 50 times. In some aspects, the polypeptide, nucleic acid molecule, vector, or recombinant virus particle described herein may reverse the inhibition of synapse generation.

[0099] As described elsewhere in this specification, in any one of the methods provided herein, a polypeptide comprising an amino acid sequence of a TAFA protein or a fragment thereof, or a variant thereof, a nucleic acid molecule encoding said polypeptide, a vector comprising said nucleic acid molecule, or a recombinant virus particle comprising said vector and a capsid protein may be administered to a subject by any suitable administration route known in the art. In some aspects, said polypeptide (or nucleic acid molecule, vector, or recombinant virus particle) is administered to a subject via intracochlear injection, vestibular injection, semicircular canal administration, tympanic membrane administration, intravenous administration, nasal administration, or a combination thereof. In some aspects, said polypeptide, nucleic acid molecule, vector, or recombinant virus particle is administered to a subject via intracochlear injection. In some aspects, said polypeptide, nucleic acid molecule, vector, or recombinant virus particle is administered to a subject via vestibular injection. In some aspects, the polypeptide, nucleic acid molecule, vector, or recombinant virus particle is administered to a subject via intratympanic or semicircular canal administration. In some aspects, the polypeptide, nucleic acid molecule, vector, or recombinant virus particle is administered to a subject via intravenous administration.

[0100] In some aspects, any of the methods provided herein (e.g., provided above) may further include administering an additional therapeutic agent to a subject. For example, some aspects of the invention include treating an inner ear disease in a subject requiring such treatment by administering a polypeptide, nucleic acid molecule, vector, or recombinant viral particle and an additional therapeutic agent to the subject. Non-limiting examples of such additional therapeutic agents are known in the art (e.g., standard treatments, such as steroids for the treatment of sudden hearing loss). The dosage and administration of one or more additional therapeutic agents are known in the art, for example, as indicated on the product description label of each drug.

[0101] When the above polypeptide, nucleic acid molecule, vector, or recombinant virus particle is administered to a subject together with an additional therapeutic agent, in some aspects, the polypeptide, nucleic acid molecule, vector, or recombinant virus particle and the additional therapeutic agent may be administered to the subject simultaneously. For example, in some aspects, the polypeptide, nucleic acid molecule, vector, or recombinant virus particle and the additional therapeutic agent may be administered to the subject as a single composition. In some aspects, the polypeptide, nucleic acid molecule, vector, or recombinant virus particle and the additional therapeutic agent may be administered simultaneously but as separate compositions. In some aspects, the polypeptide, nucleic acid molecule, vector, or recombinant virus particle and the additional therapeutic agent may be administered to the subject sequentially. For example, in some aspects, the polypeptide, nucleic acid molecule, vector, or recombinant virus particle is administered to the subject prior to the administration of the additional therapeutic agent. In some aspects, the polypeptide, nucleic acid molecule, vector, or recombinant virus particle is administered to the subject after the administration of the additional therapeutic agent.

[0102] "Polypeptide" means a chain comprising at least two consecutively linked amino acid residues, and there is no upper limit to the length of the chain. One or more amino acid residues within the protein may include modifications such as glycosylation, phosphorylation, or the formation of disulfide bonds, but are not limited thereto. "Protein" may comprise one or more polypeptides. For example, the polypeptide of the present invention may be a fusion polypeptide or fusion protein in which a second polypeptide is fused to the N-terminus or C-terminus of a polypeptide comprising the amino acid sequence of the TAFA protein or a fragment thereof, or a variant thereof. The second polypeptide may be connected directly or by a linker. The second polypeptide may be linked to a therapeutic drug of a different type from the TAFA protein or its fragment, a sequence for extending half-life including Fc or albumin, or various polypeptides for other purposes.

[0103] As used herein, the terms "nucleic acid" or "nucleic acid molecule" are intended to include DNA molecules and RNA molecules. Nucleic acid molecules may be single-stranded or double-stranded and may be cDNA.

[0104] As used herein, the term "vector" is intended to refer to a nucleic acid molecule capable of transporting another nucleic acid connected to itself. One type of vector is a "plasmid," which refers to a circular double-stranded DNA loop to which additional DNA segments can be connected. Another type of vector is a viral vector, in which case additional DNA segments can be connected to the viral genome. Certain vectors (e.g., bacterial vectors derived from replicating bacteria and episomal mammalian vectors) are capable of autoreplication within the host cell into which they are introduced. Other vectors (e.g., non-episosomal mammalian vectors) can be incorporated into the host cell's genome when introduced into a host cell and are consequently replicated along with the host genome. Additionally, certain vectors can direct the expression of genes to which they are operatively connected. Such vectors are referred to herein as "recombinant expression vectors" (or simply "expression vectors"). Generally, expression vectors useful for recombinant DNA technology are often in the form of plasmids. Since plasmids are the most commonly used form of vector, "plasmid" and "vector" may be used interchangeably in this specification. However, other forms of expression vectors that provide equivalent functions, such as viral vectors (e.g., replication-deficient retroviruses, adenoviruses, and adeno-associated viruses), are also included.

[0105] As used herein, the term "recombinant host cell" (or simply "host cell") is intended to refer to a cell containing nucleic acids that are not naturally present within the cell, and may be a cell into which a recombinant expression vector has been introduced. These terms should be understood to refer not only to the cells of a specific target but also to the offspring of such cells. Because specific modifications may occur in subsequent generations due to mutations or environmental influences, these offspring may not actually be identical to the parent cell, but are still included within the scope of the term "host cell" as used herein.

[0106] In this specification, “administration” means physically introducing a molecule (e.g., a polypeptide comprising the amino acid sequence of a TAFA protein or a fragment thereof, or a variant thereof) or a composition comprising such molecule into a subject using any of the various methods and delivery systems known to those skilled in the art. Non-limiting examples of available administration routes include, for example, intravenous, intraperitoneal, intramuscular, subcutaneous, spinal, or other parenteral administration routes by injection or infusion. As used herein, the phrase “parenteral administration” generally means a mode of administration other than enteral and local administration by injection, and includes, but is not limited to, intravenous, intraperitoneal, intramuscular, intra-arterial, intrathecal, intralymphatic, intralesional, intracapsular, intraorbital, intracardiac, intradermal, transtracheal, subcutaneous, subepidermal, intra-articular, subcapsular, subarachnoid, intraspinal, epidural, and intrasternal injections and infusions, and in vivo electroporation. The above administration may be implemented through intracochlear injection, vestibular injection, intravenous administration, tympanic membrane administration, semicircular canal administration, or a combination thereof.

[0107] As used herein, the term “object” includes any human or non-human animal. The term “non-human animal” includes all vertebrates, such as mammals and non-mammals, such as, for example, non-human primates, sheep, dogs, cattle, chickens, amphibians, reptiles, etc.

[0108] As used herein, the terms “treat,” “to treat,” and “treatment” refer to any type of intervention or process performed on a subject for the purpose of reversing, alleviating, improving, suppressing, delaying, or preventing the progression, development, severity, or recurrence of symptoms, complications, conditions, or biochemical indicators associated with a disease, or the administration of an active ingredient to a subject. Treatment may be performed on a subject with a disease or on a subject without a disease (e.g., for prevention).

[0109]

[0110] II. Polypeptide

[0111] According to one aspect of the present invention, the present invention provides a polypeptide comprising the amino acid sequence of a TAFA (TAFA Chemokine Like Family Members) protein, a fragment thereof, or a variant thereof.

[0112] According to a preferred embodiment of the present invention, the polypeptide comprises an amino acid sequence of a TAFA (TAFA Chemokine Like Family Member) protein, a fragment thereof, or a variant thereof.

[0113] According to a preferred embodiment of the present invention, the TAFA protein is one or more proteins selected from the group consisting of TAFA1 to TAFA4 proteins.

[0114] According to a preferred embodiment of the present invention, the polypeptide may be a pharmaceutical composition characterized by comprising the amino acid sequence (from the N-terminus to the C-terminus) of the following general formula 7:

[0115] <General Formula 7>

[0116] X1-X2-X3-GTCEV-X4-A-X5-H-X6- CCN-X7-N-X8-IEE- -X20-X21-X22-WWC-X23-M-X24-PC-X25-X26-GE-X27-CK-X28-LPD-X29-X30-GW-X31-C-X32-X33-G-X34-K-X35-KTT-X36-X37-X38-X39

[0117] In the above general formula 7,

[0118] X1 does not exist, or is V, I, or L,

[0119] X2 is K, E, R, or Q, and

[0120] X3 is G, T, Q, P or A, and

[0121] X4 is V or I, and

[0122] X5 is A, L, V or I, and

[0123] X6 is R or L, and

[0124] X7 is K, R, or Q, and

[0125] X8 is R or K, and

[0126] X9 is R or L, and

[0127] X10 is V or G, and

[0128] X11 is K or N, and

[0129] X12 is F or L, and

[0130] X13 is P or S, and

[0131] X14 is Q or K, and

[0132] X15 is R, H, or Q, and

[0133] X16 is A, N, S, or T, and

[0134] X17 is A, Q, R, K or T, and

[0135] X18 is D or E, and

[0136] X19 is S or A, and

[0137] X20 is I, E, L, A, or V, and

[0138] X21 is Q, G, or E, and

[0139] X22 is K or R, and

[0140] X23 is H, Q, or E, and

[0141] X24 is E, Q, N, D, S or H, and

[0142] X25 is L, V, or M, and

[0143] X26 is E, D, P, L or A, and

[0144] X27 is E or D, and

[0145] X28 is V, T, A or I, and

[0146] X29 is L, N, R, Y, S or Q, and

[0147] X30 is S, K, or T, and

[0148] X31 is S or M, and

[0149] X32 is S, A, or Y, and

[0150] X33 is S, T, or R, and

[0151] X34 is N or H, and

[0152] X35 is V or I,

[0153] X36 is R or K, and

[0154] X37 does not exist or is V, A, G, M, or N,

[0155] X38 does not exist or is T, I, N, F, or S, and

[0156] X39 is either non-existent or R, H, V, K, I, or Q.

[0157] The polypeptide comprising the amino acid sequence of the above general formula 7 may include one or more amino acid sequences selected from the group consisting of amino acid sequences presented in SEQ ID NOs 87 to 141.

[0158] According to a preferred embodiment of the present invention, a polypeptide comprising the amino acid sequence of the TAFA protein, a fragment thereof, or a variant thereof may include a sequence having at least 50% sequence identity with the amino acid sequence of SEQ ID NO. 1 or the amino acid sequence of SEQ ID NO. 15.

[0159] The polypeptide comprising the amino acid sequence of the TAFA protein, a fragment thereof, or a variant thereof may consist of an amino acid sequence of 8 to 61 amino acids, preferably 8 to 43 amino acid sequences, and more preferably 15 to 46 amino acid sequences.

[0160] A polypeptide comprising the amino acid sequence of the above TAFA protein, a fragment thereof, or a variant thereof may comprise the amino acid sequence of the following general formula 1 (from the N-terminus to the C-terminus):

[0161] <General Formula 1>

[0162] X1-GE-X2-CK-X3-L

[0163] In the above general formula 1

[0164] X1 is E, D, P, L or A, and

[0165] X2 is D or E, and

[0166] X3 is T, V, I, or A.

[0167] A polypeptide comprising the amino acid sequence of General Formula 1 above may include the amino acid sequence of SEQ ID NO. 142. The polypeptide may include an amino acid sequence selected from the group consisting of SEQ ID NOs. 1 to 14. The polypeptide may be composed of 8 to 43 amino acid sequences. The polypeptide may be composed 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 sequences.

[0168] In addition, a polypeptide comprising the amino acid sequence of the TAFA protein, a fragment thereof, or a variant thereof may comprise the amino acid sequence of the following general formula 2 (from the N-terminus to the C-terminus):

[0169] <General Formula 2>

[0170] IV-X4-X5-X6-WWC-X7-M-X8-PC-X9-X1-GE-X2-CK-X3-L

[0171] In the above general formula 2

[0172] X1 is E, D, P, L or A, and

[0173] X2 is D or E, and

[0174] X3 is T, V, I or A, and

[0175] X4 is I, E, A, L or V, and

[0176] X5 is Q, E, or G, and

[0177] X6 is K or R, and

[0178] X7 is E, H, or Q, and

[0179] X8 is E, Q, N, D, S or H, and

[0180] X9 is L, M, or V.

[0181] A polypeptide comprising the amino acid sequence of the above general formula 2 may include the amino acid sequence of SEQ ID NO. 143. The polypeptide may include an amino acid sequence selected from the group consisting of SEQ ID NOs. 28 to 51.

[0182] In addition, a polypeptide comprising the amino acid sequence of the TAFA protein, a fragment thereof, or a variant thereof may comprise the amino acid sequence of the following general formula 3 (from the N-terminus to the C-terminus):

[0183] <General Formula 3>

[0184] X1-GE-X2-CK-X3-LPD-X4-X5-GWSCS-X6-GNK-X7-KTTKVTR

[0185] In the above general formula 3

[0186] X1 is E, D, P, L or A, and

[0187] X2 is D or E, and

[0188] X3 is T, V, I or A, and

[0189] X4 is Y, S, or L, and

[0190] X5 is S or T, and

[0191] X6 is S or T, and

[0192] X7 is V or I.

[0193] The polypeptide comprising the amino acid sequence of General Formula 3 above may include the amino acid sequence of Sequence No. 144. The polypeptide may include an amino acid sequence selected from the group consisting of Sequence Nos. 52 to 58.

[0194] In addition, a polypeptide comprising the amino acid sequence of the TAFA protein, a fragment thereof, or a variant thereof may comprise the amino acid sequence of the following general formula 4 (from the N-terminus to the C-terminus):

[0195] <General Formula 4>

[0196] G-X1-VAGTT-X2-X3-X4-PSCV-X5-X6-X7

[0197] In the above general formula 4

[0198] X1 is Q or K, and

[0199] X2 is R, H, or Q, and

[0200] X3 is A, N, S or T, and

[0201] X4 is R, A, Q, K, or T, and

[0202] X5 is D or E, and

[0203] X6 is A or does not exist, and

[0204] X7 is S, A, or does not exist.

[0205] A polypeptide comprising the amino acid sequence of General Formula 4 above may include the amino acid sequence of SEQ ID NO. 145. The polypeptide may include an amino acid sequence selected from the group consisting of SEQ ID NOs 15 to 27. The polypeptide may be composed of 15 to 46 amino acid sequences. The amino acid sequence of the polypeptide may be composed 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 sequences.

[0206] In addition, a polypeptide comprising the amino acid sequence of the TAFA protein, a fragment thereof, or a variant thereof may comprise the amino acid sequence of the following general formula 5 (from the N-terminus to the C-terminus):

[0207] <General Formula 5>

[0208] X8-IEE-X9-SQT-X10-X11-CSC-X12-X13-G-X1-VAGTT-X2-X3-X4-PSCV-X5-X6-X7

[0209] In the above general formula 5

[0210] X1 is Q or K, and

[0211] X2 is R, H, or Q, and

[0212] X3 is A, N, S or T, and

[0213] X4 is R, A, Q, K, or T, and

[0214] X5 is D or E, and

[0215] X6 is A or does not exist,

[0216] X7 is S, A, or does not exist,

[0217] X8 is R or K, and

[0218] X9 is R or L, and

[0219] X10 is V or G, and

[0220] X11 is K or N, and

[0221] X12 is F or L, and

[0222] X13 is P or S.

[0223] A polypeptide comprising the amino acid sequence of the above general formula 5 may include the amino acid sequence of SEQ ID NO. 146. The polypeptide may include an amino acid sequence selected from the group consisting of SEQ ID NOs 59 to 74.

[0224] In addition, a polypeptide comprising the amino acid sequence of the TAFA protein, a fragment thereof, or a variant thereof may comprise the amino acid sequence of the following general formula 6 (from the N-terminus to the C-terminus):

[0225] <General Formula 6>

[0226] G-X1-VAGTT-X2-X3-X4-PSCV-X5-X6-X7-IV-X8-X9-KWWC-X10-M-X11-PC-X12

[0227] In the above general formula 6

[0228] X1 is Q or K, and

[0229] X2 is R, H, or Q, and

[0230] X3 is A, N, S or T, and

[0231] X4 is R, A, Q, K or T, and

[0232] X5 is D or E, and

[0233] X6 is A or does not exist,

[0234] X7 is S, A, or does not exist,

[0235] X8 is I, A, V or L, and

[0236] X9 is Q or E, and

[0237] X10 is H or Q, and

[0238] X11 is N, D, S or H, and

[0239] X12 is L or M.

[0240] A polypeptide comprising the amino acid sequence of the above general formula 6 may include the amino acid sequence of SEQ ID NO. 147. The polypeptide may include an amino acid sequence selected from the group consisting of SEQ ID NOs 75 to 85.

[0241] A polypeptide comprising an amino acid sequence selected from the group consisting of general formulas 1 to 6 above may include one or more amino acid sequences selected from the group consisting of amino acid sequences presented in SEQ ID NOs 142 to 147. The polypeptide may include an amino acid sequence selected from the group consisting of SEQ ID NOs 1 to 85.

[0242]

[0243] III. Nucleic Acids

[0244] According to another aspect of the present invention, the present invention provides a nucleic acid molecule encoding the polypeptide.

[0245]

[0246] The above polypeptide, TAFA protein, or fragment thereof or variant thereof is as described in II.

[0247] Nucleic acid molecules useful for the present disclosure are not particularly limited as long as said nucleic acid molecules can be translated into polypeptides upon transduction into cells. In some embodiments, said nucleic acid molecules code for polypeptides (e.g., TAFA polypeptides or polypeptides comprising any one of the amino acid sequences of SEQ ID NOs 142 to 147) or fusion proteins.

[0248] In some embodiments, the nucleic acid codes for a protein useful for the prevention or treatment of a disease or disorder such as those described herein. In some embodiments, the nucleic acid codes for a peptide for the prevention or treatment of a specific disease intended for sustained expression within the body of a subject or patient.

[0249] 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 molecule encoding the TAFA protein or a fragment thereof or a variant thereof.

[0250] In some embodiments, the nucleic acid encoding the polypeptide further comprises a sequence encoding a signal sequence.

[0251] In some embodiments, the polynucleotide described herein further comprises a regulatory element. Accordingly, in some embodiments, the polynucleotide comprises (1) a regulatory element, (2) a non-translating nucleic acid sequence described herein (e.g., an EF-1α intron or a fragment thereof), and (3) a foreign gene (e.g., a nucleic acid molecule encoding a polypeptide comprising any one of the amino acid sequences of SEQ ID NOs 142 to 147).

[0252] As used herein, the term “regulatory element” refers to a nucleic acid sequence that regulates (e.g., increases or decreases) the expression of operably linked nucleic acids. A regulatory element useful for the present disclosure comprises an enhancer (e.g., a CMV enhancer), a promoter (e.g., a CMV promoter, an EF-1α promoter, or a β-actin promoter), an exon (e.g., exon 1 or exon 2), a splicing donor sequence, a receptor sequence, or a combination thereof. In some embodiments, the regulatory element may comprise a sequence for transcription termination (e.g., a poly A), a sequence for stably expressing a foreign gene (e.g., a WPRE sequence), a sequence for reducing the occurrence of foreign gene-specific immunity (e.g., a miRNA target sequence), or a combination thereof.

[0253]

[0254] IV. Vectors containing nucleic acids encoding polypeptides

[0255] According to another aspect of the present invention, the present invention provides a vector comprising the nucleic acid molecule.

[0256]

[0257] The above nucleic acid molecule is as described in III.

[0258] As described herein, such vectors are useful for recombinant expression in host cells and cells targeting therapeutic interventions. In some embodiments, vectors useful for the delivery of polynucleotides described herein (e.g., TAFA polypeptides or nucleic acid molecules encoding polypeptides comprising any one of the amino acid sequences of SEQ ID NOs. 142 to 147) include viral vectors. Examples of viruses that may be used as vectors in this disclosure include, but are not limited to, retroviruses, herpes simplex viruses, lentiviruses, poxviruses, vaccinia viruses, rhabdoviruses, adenoviruses, helper-dependent adenoviruses, adeno-associated viruses (AAVs), baculoviruses, and combinations thereof. In some embodiments, vectors that may be used in this disclosure include non-viral vectors. Non-limiting examples of such vectors include plasmids, cosmids, yeast artificial chromosomes (YACs), bacteriophages, and combinations thereof.

[0259] In some embodiments, the vector further comprises one or more sequences selected from the group consisting of a promoter sequence, an enhancer sequence, an exon sequence, an intron sequence, a signal sequence coding sequence, a splicing donor sequence, and one or more adeno-associated virus inverted terminal repeat (ITR) sequences.

[0260]

[0261] According to another aspect of the present invention, the present invention provides a recombinant virus particle comprising the vector and the capsid protein.

[0262]

[0263] According to a preferred embodiment of the present invention, the virus may be an AAV.

[0264]

[0265] V. AAV

[0266] In some embodiments, the polynucleotide described herein (e.g., a TAFA polypeptide or a nucleic acid molecule encoding a polypeptide comprising any one of the amino acid sequences of SEQ ID NOs 142 to 147) is delivered to a cell, for example, using an AAV. As a single-stranded DNA virus, the adeno-associated virus (AAV) is a helper-dependent human parvovirus. The AAV genome has a size of about 4.7 kbp and consists of an N-terminus encoding the rep gene involved in viral replication and the expression of viral genes, a C-terminus encoding the cap gene encoding the capsid protein of said virus, and an inverse repeat end (ITR) with about 145 bases inserted into each end. The 145 bp inverse repeat end (ITR) has a T-shaped structure, functions as a replication origin during viral genome replication, and acts as a primary packaging signal. The above ITR is the only cis-acting sequence required when constructing a recombinant AAV (rAAV) construct. The above IRT exhibits enhancer activity in the presence of the Rep protein, but has very weak activity in the absence of the Rep protein. When cloning foreign genes into a recombinant AAV construct, these characteristics are taken into account to appropriately configure the enhancer, promoter, pA, etc., to construct an expression construct (RJ Samulski and N Muzyczka, Annu. Rev. Virolo. 2014. 1:427-451). Four proteins are translated from the rep gene. These proteins are classified as rep78, rep68, rep52, and rep40 according to their molecular weight and perform important functions in AAV DNA replication. Four proteins are translated from the cap gene. Among these, VP1, VP2, and VP3 proteins are structural proteins that constitute AAV particles, and assembly-activating protein (AAP) promotes the assembly of AAV particles by said structural proteins.For the efficient replication of adeno-associated viruses, some proteins and RNAs derived from helper viruses such as adenoviruses or herpes simplex viruses are required (Muzyczka N. Curr Top Microbiol Immunol 158, 97-129, 1992).

[0267] AAV possesses unique characteristics that make it attractive as a vector for delivering foreign genes (transgenes) to cells. AAV infection of cells in cultures has generally been non-cellular, and natural infections in humans and other animals are asymptomatic. Furthermore, AAV has the potential to target many different tissues in vivo by infecting many different types of mammalian cells. AAV also has additional advantages that make it a particularly attractive viral system for gene delivery, including promoting a weak immune response compared to other forms of gene delivery and sustaining expression based on non-integrating, episomal vector DNA in both dividing and quiescent cells. Additionally, since AAV withstands the conditions used to inactivate adenoviruses (56°C to 65°C for several hours), cryopreservation of rAAV-based vaccines becomes less critical.

[0268] The type of 아데노-연관 virus that can be used in this introduction is AAVrh.10(AAVrh10), AAV-DJ(AAVDJ), AAV-DJ8(AAVDJ8), AAV1, AAV2, AAV2G9, AAV3, AAV3a, AAV3b, AAV3-3, AAV4, AAV4-4, AAV5, AAV6, AAV6.1, AAV6.2, AAV6.1.2, AAV7, AAV7.2, AAV8, AAV9, AAV9.11, AAV9.13, AAV9.16, AAV9.24, AAV9.45, AAV9.47, AAV9.61, AAV9.68, AAV9.84, AAV9.9, AAV10, AAV11, AAV12, AAV16.3, AAV24.1, AAV27.3, AAV42.12, AAV42-1b, AAV42-2, AAV42-3a, AAV42-3b, AAV42-4, AAV42-5a, AAV42-5b, AAV42-6b, AAV42-8, AAV42-10, AAV42-11, AAV42-12, AAV42-13, AAV42-15, AAV42-aa, AAV43-1, AAV43-12, AAV43-20, AAV43-21, AAV43-23, AAV43-25, AAV43-5, AAV44.1, AAV44.2, AAV44.5, AAV223.1, AAV223.2, AAV223.4, AAV223.5, AAV223.6, AAV223.7, AAV1-7 / rh.48, AAV1-8 / rh.49, AAV2-15 / rh.62, AAV2-3 / rh.61, AAV2-4 / rh.50, AAV2-5 / rh.51, AAV3.1 / hu.6, AAV3.1 / rh.9, AAV3-9 / rh.52, AAV3-11 / rh.53, AAV4-8 / r11.64, AAV4-9 / rh.54, AAV4-19 / rh.55, AAV5-3 / rh.57, AAV5-22 / rh.58, AAV7.3 / hu.7, AAV16.8 / hu.10, AAV16.12 / hu.11, AAV29.3 / bb.1, AAV29.5 / bb.2, AAV106.1 / hu.37, AAV114.3 / hu.40, AAV127.2 / hu.41, AAV127.5 / hu.42, AAV128.3 / hu.44, AAV130.4 / hu.48, AAV145.1 / hu.53, AAV145.5 / hu.54, AAV145.6 / hu.55, AAV161.10 / hu.60, AAV161.6 / hu.61, AAV33.12 / hu.17, AAV33.4 / hu.15, AAV33.8 / hu.16, AAV52 / hu.19, AAV52.1 / hu.20, AAV58.2 / hu.25, AAVA3.3, AAVA3.4, AAVA3.5, AAVA3.7, AAVC1, AAVC2, AAVC5, AAVF3, AAVF5, AAVH2, AAVrh.72, AAVhu.8, AAVrh.68, AAVrh.70, AAVpi.1, AAVpi.3, AAVpi.2, AAVrh.60, AAVrh.44, AAVrh.65, AAVrh.55, AAVrh.47, AAVrh.69, AAVrh.45, AAVrh.59, AAVhu.12, AAVH6, AAVH03, AAVH-1 / hu.1, AAVH-5 / hu.3, AAVLG-10 / rh.40, AAVLG-4 / rh.38, AAVLG-9 / hu.39, AAVN721-8 / rh.43, AAVCh.5, AAVCh.5R1, AAVcy.2, AAVcy.3, AAVcy.4, AAVcy.5, AAVCy.5R1, AAVCy.5R2, AAVcy.5R3, AAVCy.5R4, AAVcy.6, AAVhu.1, AAVhu.2, AAVhu.3, AAVhu.4, AAVhu.5, AAVhu.6, AAVhu.7, AAVhu.9, AAVhu.10, AAVhu.11, AAVhu.13, AAVhu.15, AAVhu.16, AAVhu.17, AAVhu.18, AAVhu.20, AAVhu.21, AAVhu.22, AAVhu.23.2, AAVhu.24, AAVhu.25, AAVhu.27, AAVhu.28, AAVhu.29, AAVhu.29R, AAVhu.31, AAVhu.32, AAVhu.34, AAVhu.35, AAVhu.37, AAVhu.39, AAVhu.40, AAVhu.41, AAVhu.42, AAVhu.43, AAVhu.44, AAVhu.44R1, AAVhu.44R2, AAVhu.44R3, AAVhu.45, AAVhu.46, AAVhu.47, AAVhu.48, AAVhu.48R1, AAVhu.48R2, AAVhu.48R3, AAVhu.49, AAVhu.51, AAVhu.52, AAVhu.54, AAVhu.55, AAVhu.56, AAVhu.57, AAVhu.58, AAVhu.60, AAVhu.61, AAVhu.63, AAVhu.64, AAVhu.66, AAVhu.67, AAVhu.14 / 9, AAVhu.t19, AAVrh.2, AAVrh.2R. AAVrh.8, AAVrh.8R, AAVrh.12, AAVrh.13, AAVrh.13R, AAVrh.14, AAVrh.17, AAVrh.18, AAVrh.19, AAVrh.20, AAVrh.21, AAVrh.22, AAVrh.23, AAVrh.24, AAVrh.25, AAVrh.31, AAVrh.32, AAVrh.33, AAVrh.34, AAVrh.35, AAVrh.36, AAVrh.37, AAVrh.37R2, AAVrh.38, AAVrh.39, AAVrh.40, AAVrh.46, AAVrh.48, AAVrh.48.1, AAVrh.48.1.2, AAVrh.48.2, AAVrh.49, AAVrh.51, AAVrh.52, AAVrh.53, AAVrh.54, AAVrh.56, AAVrh.57, AAVrh.58, AAVrh.61, AAVrh.64, AAVrh.64R1, AAVrh.64R2, AAVrh.67, AAVrh.73, AAVrh.74, AAVrh8R. AAVrh8R A586R 변이체, AAVrh8R R533A 변이체, AAAV, BAAV, 염소 AAV, 소 AAV, AAVhE1.1, AAVhEr1.5, AAVhEr1.14, AAVhEr1.14, AAVhEr1.8, AAVhEr1.16, AAVhEr1.18, AAVhEr1.35, AAVhEr1.7, AAVhEr1.36, AAVhEr2.29, AAVhEr2.4, AAVhEr2.16, AAVhEr2.16, AAVhEr2.30, AAVhEr2.31, AAVhEr2.31, AAVhEr2.36, AAVhER1.23, AAVhEr3.1, AAV2.5T, AAV-PAEC, AAV-LK01, AAV-LK02, AAV-LK03, AAV-LK04, AAV-LK05, AAV-LK06, AAV-LK07, AAV-LK08, AAV-LK09, AAV-LK10, AAV-LK11, AAV-LK12, AAV-LK13, AAV-LK14, AAV-LK15, AAV-LK16, AAV-LK17, AAV-LK18, AAV-LK19, AAV-PAEC2, AAV-PAEC4, AAV-PAEC6, AAV-PAEC7, AAV-PAEC8, AAV-PAEC11, AAV-PAEC12, AAV-2-pre-miRNA-101, AAV-8h, AAV-8b, AAV-h, AAV-b, AAV SM 10-2, AAV Shuffle 100-1, AAV Shuffle 100-3, AAV Shuffle 100-7, AAV Shuffle 10-2, AAV Shuffle 10-6, AAV Shuffle 10-8, AAV Shuffle 100-2, AAV SM 10-1, AAV SM 10-8, AAV SM 100-3, AAV SM 100-10, B P61 AAV, B P62 AAV, B P63 AAV, AAVrh.50, AAVrh.43, AAVrh.62, AAVrh.48, AAVhu.19, AAVhu.11, AAVhu.53, AAV4-8 / rh.64, AAVLG-9 / hu.39, AAV54.5 / hu.23, AAV54.2 / hu.22, AAV54.7 / hu.24, AAV54.1 / hu.21, AAV54.4R / hu.27, AAV46.2 / hu.28, AAV46.6 / hu.29, AAV128.1 / hu.43, true type AAV(ttAAV), UPENN AAV 10 and Japanese AAV 10 serotypes, but not limited thereto.

[0269] In some embodiments, the serotype of the adeno-associated virus is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, or AAVrh10. In some embodiments, the serotype of the AAV is AAV2. In some embodiments, the serotype of the AAV is AAV5. In some embodiments, the serotype of the AAV is AAV8. In some embodiments, the serotype of the AAV is AAV9.

[0270] In some embodiments, for the efficient expression or production of the adeno-associated virus, or for other purposes such as targeting, some amino acid sequences of the Rep protein and / or Cap protein may be mutated, or new amino acid sequences may be added or removed, and the sequence of the gene encoding them may also be mutated. The mutated AAV is also included in the AAV of the present invention insofar as it functions as an AAV.

[0271]

[0272] VI. Cells

[0273] In some embodiments, the present specification provides a cell comprising any of the polynucleotides described herein (e.g., a TAFA polypeptide or a nucleic acid molecule encoding a polypeptide comprising any one of the amino acid sequences of SEQ ID NOs 142 to 147) or a cell comprising a vector comprising said polynucleotide.

[0274] For example, in some embodiments, the cell is transfected, transfected, or transformed using a vector (e.g., an AAV vector) comprising a foreign gene described in this specification (e.g., a nucleic acid molecule encoding a polypeptide comprising any one of the amino acid sequences of SEQ ID NOs 142 to 147) and a non-coding nucleic acid sequence for foreign gene expression.

[0275] Although not bound by any single theory, in some embodiments, the cells described herein (e.g., transduced with a polynucleotide containing a non-coding nucleic acid sequence) are useful for producing proteins such as those encoded by the foreign gene described herein (e.g., a TAFA polypeptide or a polypeptide containing any one of the amino acid sequences of SEQ ID NOs 142 to 147). As described herein, in some embodiments, the non-coding nucleic acid sequence described herein (e.g., an EF-1α intron or a fragment thereof) can enhance the expression of the protein encoded by said foreign gene ("coded protein") in the cells. Thus, in some embodiments, the cells described herein (e.g. transduced with a polynucleotide containing the foreign gene and the non-coding nucleic acid sequence of the present disclosure) produce greater expression of said coded protein compared to a reference cell. In some embodiments, the reference cell is transduced with the corresponding polynucleotide but lacks a non-coding nucleic acid sequence.

[0276] In some embodiments, the cells described herein may produce the protein encoded by the foreign gene in vitro. In certain embodiments, the cells described herein may produce the encoded protein in vivo (e.g., in a subject administered the polynucleotide described herein). In some embodiments, the cells described herein may produce the encoded protein both in vitro and in vivo.

[0277] In some embodiments, the cell that can be used to produce a protein encoded by a foreign gene (e.g., in vitro) includes a host cell. As used herein, the term “host cell” is intended to include any cell of an organism that is transfected with the expression construct or vector (e.g., an AAV vector) and is capable of replicating the expression construct or expressing the gene encoded by the expression construct. Such cells include eukaryotic cells and prokaryotic cells. As used herein, the term “transfection” is intended to include transfection and transformation. The host cell may be transfected, transfected, or transformed by the expression construct. This process means that an exogenous nucleic acid molecule is delivered or introduced into the host cell. In some embodiments, the host cell is an isolated host cell containing the AAV vector. In some embodiments, the host cell is an isolated host cell transformed with the AAV vector.

[0278] In some embodiments, the host cell is a eukaryotic cell. In some embodiments, the host cell is selected from the group consisting of mammalian cells, insect cells, yeast cells, transgenic mammalian cells, and plant cells. In some embodiments, the host cell is a prokaryotic cell. In some embodiments, the prokaryotic cell is a bacterial cell.

[0279] In some embodiments, the host cell is an insect cell. In some embodiments, the insect cell is Sf9. In some embodiments, the host cell is a mammalian cell. Non-limiting examples of mammalian cells available for use in this disclosure include HEK293, or cells derived from HEK293 cells (293T, 293FT, 293F / FreeStyle 293-F, 293E, Expi293 / Expi293F, HEK293S, HEK293A / 293H, 293T / 17, etc.), 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, Includes 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.

[0280] In some embodiments, the cell that can be used to produce a protein encoded by the foreign gene described herein (e.g. in vivo) comprises a human cell. In some embodiments, the human cell is a cell of a subject that has been administered the nucleic acid molecule described herein. In certain embodiments, the human cell is derived from a donor (e.g., a healthy human subject).

[0281] In some embodiments, the present disclosure provides a composition comprising an AAV vector or a host cell comprising said AAV vector or said AAV vector or said AAV vector transformed into said AAV vector.

[0282]

[0283] VII. Composition

[0284] According to another aspect of the present invention, the present invention provides a composition comprising the polypeptide, a nucleic acid molecule encoding the polypeptide, a vector comprising the nucleic acid molecule, a recombinant virus particle comprising the vector and a capsid protein, a cell comprising the vector, a cell transformed with the vector, or a combination thereof.

[0285]

[0286] The above polypeptides, nucleic acid molecules, vectors, recombinant virus particles, and cells are as described in II to VI.

[0287] In some embodiments, the composition is a pharmaceutical composition.

[0288] In some embodiments, the present specification discloses a pharmaceutical composition 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 NOs 142 to 147) or a nucleic acid molecule encoding the same, and (b) one or more pharmaceutically acceptable carriers. In some embodiments, the present specification discloses a pharmaceutical composition comprising (a) a vector described herein (e.g., rAAV) or a recombinant virus particle, and (b) one or more pharmaceutically acceptable carriers. In some embodiments, the present specification discloses a pharmaceutical composition comprising (a) a cell described herein and (b) one or more pharmaceutically acceptable carriers.

[0289] In some embodiments, the pharmaceutical composition described herein comprises a polypeptide consisting of a sequence of 8 to 61 amino acids.

[0290] In some embodiments, the pharmaceutical composition described herein comprises a polypeptide 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.

[0291] In some embodiments, the pharmaceutical composition is a pharmaceutical composition for the prevention, improvement, or treatment of inner ear diseases.

[0292] In some embodiments, the inner ear disease is caused by damage or abnormality of the auditory system or the balance system (vestibular system) in the inner ear. In some embodiments, the inner ear disease includes two types of organs in which ribbon synapses exist within the inner ear, namely, i) auditory system disease or dysfunction in the cochlea (inner hair cell (IHC) and outer hair cell (OHC)) and ii) balance system (vestibular system) disease or dysfunction (vestibular synaptopathy) in the vestibular organs (oval capsule, saccule, and semicircular canals).

[0293] The above auditory disorders or dysfunctions include, but are not limited to, hidden hearing loss, noise-induced synaptopathy, age-related synaptopathy, tinnitus, sensorineural hearing loss (SNHL), or combinations thereof.

[0294] The above balance system (vestibular system) disorders or dysfunctions include, but are not limited to, chronic vestibular decline, transient vestibular syncope, acute vestibular neuropathy, age-related vestibular decline, autoimmune inner ear disease, auditory neuropathy, or combinations thereof.

[0295] In some embodiments, the inner ear disease is caused by damage to the spiral ganglion itself, degeneration of the neurites, or a disorder of the branching point. Diseases caused by damage to the spiral ganglion itself, degeneration of the neurites, or a disorder of the branching point include Auditory Neuropathy Spectrum Disorder (ANSD), Noise-induced hearing loss, Age-related hearing loss (ARHL), diabetic auditory neuropathy, Spiral ganglionitis, vestibular schwannoma, or a combination thereof.

[0296] For the treatment of the above-mentioned inner ear disease, the vector of the present invention (e.g., rAAV) or recombinant virus particle is 1 x 10 5 Up to 1x10 13 vg / ear, preferably 1x10 6 Up to 1x10 12 vg / ear, more preferably 1x10 7 Up to 1x10 12 vg / ear, most preferably 1x10 8 Up to 1x10 11 It can be administered at a dose of vg / ear.

[0297] Pharmaceutically acceptable carriers available for use in the present disclosure are those commonly used in formulations. Examples of such pharmaceutically acceptable carriers include, but are not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia gum, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methylcellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil. The pharmaceutical compositions of the present disclosure may further include one or more additives selected from the group consisting of 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 ed., 1995).

[0298] The pharmaceutical compositions of the present disclosure are formulated to be compatible with their intended routes of administration. Examples of suitable parenteral routes of administration include intracochlear injection, intravesical injection, intracircular canal administration, intratympanic administration, intravenous administration, nasal administration, or combinations thereof. In some aspects, the polypeptide, nucleic acid molecule, vector, or recombinant virus particle is administered to a subject via intracochlear injection. In some aspects, the polypeptide, nucleic acid molecule, vector, or recombinant virus particle is administered to a subject via intravesical injection. In some aspects, the polypeptide, nucleic acid molecule, vector, or recombinant virus particle is administered to a subject via intratympanic or intracircular canal administration. In some aspects, the polypeptide, nucleic acid molecule, vector, or recombinant virus particle is administered to a subject via intravenous administration. The amino acid sequence of the polypeptide disclosed in the present invention (e.g., a TAFA protein fragment or a variant thereof) may consist of 8 to 61 amino acids, preferably 8 to 30 amino acids, so that a desired preventive or therapeutic effect can be achieved through administration via the nasal cavity.

[0299] In some embodiments, the pharmaceutical composition is administered at a daily dose of 0.0001 to 100 mg / kg.

[0300] The pharmaceutical composition of the present disclosure may be formulated with one or more pharmaceutically acceptable carriers and / or excipients. The pharmaceutical composition may be provided in a unit dosage form or dispensed into a multi-dose container. The formulation may be in the form of a solution, suspension, or emulsion in an oil or aqueous medium, or in the form of an extract, powder, granule, tablet, or capsule. The formulation may further comprise a dispersant or a stabilizer.

[0301]

[0302] VIII. Kit

[0303] The present specification also discloses a kit comprising one or more polypeptides disclosed herein (e.g., a TAFA polypeptide or a polypeptide comprising any one of the amino acid sequences of SEQ ID NOs 142 to 147), one or more polynucleotides (e.g., a foreign gene and a non-translating nucleic acid sequence), one or more vectors disclosed herein (e.g., an AAV vector), one or more cells disclosed herein (e.g., a host cell comprising the AAV vector or transformed with the AAV vector), any composition disclosed herein, or any combination thereof. In some embodiments, the kit comprises instructions for use.

[0304] The terms “kit” and “system” as used herein are intended to refer to at least one polynucleotide disclosed herein in combination with one or more other types of elements or components (e.g., other types of biochemical reagents, containers, packages such as commercial packaging, instructions for use, etc.), one or more vectors disclosed herein (e.g., AAV vectors), one or more host cells disclosed herein, any pharmaceutical composition disclosed herein, or any combination thereof.

[0305]

[0306] IX. Uses and Methods

[0307] IX.A. Production Method

[0308] The present specification also provides a method for preparing a composition comprising the step of preparing the above polypeptide (e.g., a TAFA polypeptide or a polypeptide comprising any one of the amino acid sequences of SEQ ID NOs 142 to 147), or a nucleic acid molecule encoding the above polypeptide, a vector comprising the above nucleic acid molecule, a recombinant virus particle comprising the above vector and a capsid protein, a cell comprising the above vector, a cell transformed with the above vector, or a combination thereof.

[0309] In some embodiments, this method comprises synthesizing the TAFA protein or a fragment thereof or a variant thereof described herein under suitable conditions.

[0310] In some embodiments, this method comprises culturing the cells described herein (e.g., transduced with a polynucleotide comprising a foreign gene and a non-coding nucleic acid molecule) under suitable conditions and recovering the encoded protein. In certain embodiments, a method for producing a polypeptide encoded by a foreign gene comprises administering the polynucleotide of this disclosure (e.g., comprising a foreign gene and a non-coding nucleic acid molecule) to a target in need to produce the encoded polypeptide in said target. Further disclosures regarding such in vivo methods for producing polypeptides are provided elsewhere in this disclosure (e.g., see Therapeutic Uses).

[0311] In some embodiments, the present disclosure provides a method for producing recombinant adeno-associated virus particles comprising polynucleotides (e.g., including foreign genes and non-translating nucleic acid sequences) as described herein. In some embodiments, the method for producing such recombinant AAV comprises culturing cells transfected with an AAV vector as described herein under conditions for producing recombinant AAV. In some embodiments, the method further comprises the step of isolating the produced recombinant virus particles.

[0312] In some embodiments, the present disclosure provides recombinant virus particles produced by the above method.

[0313] In some embodiments, the recombinant virus particle may be produced by a step comprising (i) transfecting a cell with an AAV vector containing the foreign gene (e.g., a TAFA polypeptide or a nucleic acid molecule encoding a polypeptide containing any one of the amino acid sequences of SEQ ID NOs 142 to 147) and (ii) a construct containing rep and cap genes. Additionally, (iii) it may be produced using a helper construct for transfecting the foreign gene into a host cell. In such embodiments, the helper construct may contain an E2A gene that promotes AAV genome replication and gene transcription, an E4 gene that enables AAV mRNA to move from the nucleus to the cytoplasm, and a VA region that generates two VA RNAs that serve to regulate translation.

[0314] In some embodiments, the three constructs described above may be replaced by two constructs for transduction into host cells. In these embodiments, the AAV construct comprises a foreign gene and a non-translating nucleic acid sequence, and a separate construct comprises the rep and cap genes, the E2A gene, the E4 gene, and the VA region. Further methods for producing the AAV particles described herein are generally known in the art. For example, see Clement et al., Mol Ther Methods Clin Dev 3: 16002 (March 2016); Clark, Kidney Int. 61: S9-15 (January 2002); and Xiao et al., J Virol 72(3): 2224-32 (March 1998), each of which is incorporated herein by reference in its entirety.

[0315] The present specification also discloses a recombinant virus particle comprising (a) a capsid protein and (b) the AAV vector.

[0316]

[0317] IX.B. Therapeutic Uses

[0318] Polypeptides described herein (e.g., TAFA polypeptide or polypeptides comprising any one of the amino acid sequences of SEQ ID NOs 142 to 147), nucleic acids (e.g., including foreign genes and non-translating nucleic acid sequences), vectors containing such nucleic acids and recombinant viruses (e.g., rAAV), cells comprising said nucleic acids or vectors, and methods described herein have many in vitro and in vivo uses. For example, polypeptides, polynucleotides, and vectors described herein, such as AAV vectors, can be administered to cells in cultures, in vitro, or in vitro, or to human subjects, e.g., in vivo, to prevent or treat diseases. Accordingly, in some embodiments, the present disclosure provides a use for the prevention or treatment of inner ear disease for the manufacture of a drug comprising a polypeptide comprising an amino acid sequence of a TAFA protein or a fragment thereof or a variant thereof described herein, 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 a combination thereof. Additionally, in some embodiments, the present disclosure provides a use for the prevention or treatment of disease caused by inner ear ribbon synapse damage for the manufacture of a drug comprising a polypeptide comprising an amino acid sequence of a TAFA protein or a fragment thereof or a variant thereof described herein, 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 a combination thereof.

[0319] In some embodiments, the TAFA protein described herein is one or more proteins selected from the group consisting of TAFA1 to TAFA4 proteins.

[0320] In some embodiments, the present specification discloses a method for expressing a foreign gene in a subject in need, comprising administering to the subject a polynucleotide disclosed herein (e.g., including a foreign gene and a non-translating nucleic acid sequence), a vector disclosed herein, a recombinant virus disclosed herein (e.g., rAAV), a cell disclosed herein, or a pharmaceutical composition disclosed herein, wherein the expression of the foreign gene in the subject is increased after administration.

[0321] As described in this specification, the non-translating nucleic acid sequence of this disclosure may increase the expression of said foreign gene when said foreign gene is translated. Accordingly, in some embodiments, this disclosure relates to a method for increasing the expression of a foreign gene in a cell, comprising contacting said cell with any polynucleotide, vector, or recombinant virus among the polynucleotides disclosed in this specification (e.g., TAFA polypeptide or a nucleic acid molecule encoding a polypeptide comprising any one of the amino acid sequences of SEQ ID NOs 142 to 147), vectors, or recombinant viruses (e.g., rAAV). said contact may be performed in vitro or in vivo. When said contact is performed in vivo, the method may further comprise administering any polynucleotide, vector, or recombinant virus among said polynucleotides, vectors, or recombinant viruses to a target prior to contact.

[0322] In some embodiments, the expression of the foreign gene (e.g., a nucleic acid molecule encoding a polypeptide comprising any one of the amino acid sequences of SEQ ID NOs 142 to 147) after contact is increased by at least about 1, at least about 1.1, at least about 1.2, at least about 1.3, at least about 1.4, at least about 1.5, at least about 1.6, at least about 1.7, at least about 1.8, at least about 1.9, at least about 2, at least about 2.5, at least about 3, at least about 3.5, at least about 4, at least about 5, at least about 6, at least about 7, at least about 8, at least about 9, or at least about 10 times or more compared to the reference expression. In some embodiments, the reference expression is the expression of the foreign gene within the cell prior to contact. In some embodiments, the reference expression is the expression of a foreign gene within the corresponding cell that has not come into contact with the polypeptide, polynucleotide, vector, or recombinant virus described herein.

[0323] Another aspect of the present disclosure provides a method for preventing or treating a disease in a subject in need, comprising administering to the subject an effective amount of any polypeptide, polynucleotide, vector, cell, recombinant virus, or pharmaceutical composition among the polypeptides (e.g., TAFA polypeptide or polypeptide comprising any one of the amino acid sequences of SEQ ID NOs 142 to 147), polynucleotides (e.g., nucleic acid molecules encoding TAFA polypeptide or polypeptide comprising any one of the amino acid sequences of SEQ ID NOs 142 to 147), vectors, cells, recombinant viruses, or pharmaceutical compositions. As is apparent from the present disclosure, the compositions described herein (e.g., polypeptides, polynucleotides, recombinant expression constructs, cells, pharmaceutical compositions, or recombinant viruses) may be used to prevent or treat any target disease.

[0324] In some embodiments, the method may further include administering an additional therapeutic agent (e.g., a hearing loss treatment, a steroid, etc.) to the subject. In some embodiments, the additional therapeutic agent may be administered to the subject simultaneously with, before, or after administration of the polypeptide, polynucleotide, vector, cell, recombinant virus, or pharmaceutical composition.

[0325] Diseases that can be prevented, improved, or treated by the present disclosure are not limited and include all diseases for which it is necessary to reduce the frequency of drug administration. Non-limiting examples of such diseases include inner ear diseases.

[0326] The above inner ear disease includes two types of organs in which ribbon synapses exist within the inner ear, namely, i) auditory system disease or dysfunction in the cochlea (inner hair cell (IHC) and outer hair cell (OHC)) and ii) vestibular system disease or dysfunction (vestibular synaptopathy) in the vestibular organs (oval capsule, saccule, and semicircular canals).

[0327] The above auditory disorders or dysfunctions include, but are not limited to, hidden hearing loss, noise-induced synaptopathy, age-related synaptopathy, tinnitus, sensorineural hearing loss (SNHL), or combinations thereof.

[0328] The above balance system (vestibular system) disorders or dysfunctions include, but are not limited to, chronic vestibular decline, transient vestibular syncope, acute vestibular neuropathy, age-related vestibular decline, autoimmune inner ear disease, auditory neuropathy, or combinations thereof.

[0329] In some embodiments, the present disclosure provides a method for reducing or inducing the formation of ribbon synapses in the inner ear of a subject in need, comprising administering to the subject a composition comprising a polypeptide having an amino acid sequence of a TAFA protein or a fragment thereof or a variant thereof, a nucleic acid molecule encoding said polypeptide, a vector having said nucleic acid molecule, a recombinant virus particle having said vector and a capsid protein, a cell having said vector, a cell transformed with said vector, or a combination thereof.

[0330] The above ribbon synapses may be present in i) the cochlea (inner hair cells (IHC) and outer hair cells (OHC)) and / or ii) the vestibular system (oval capsule, saccule, and semicircular canals).

[0331] Another aspect of the present disclosure provides a gene therapy or a method for the prevention or treatment of a disease capable of achieving sustained foreign gene expression.

[0332] In some embodiments, the present disclosure provides a method for treating an inner ear disease in a subject in need, comprising administering to the subject a composition comprising a polypeptide having an amino acid sequence of a TAFA protein or a fragment thereof or a variant thereof, a nucleic acid molecule encoding said polypeptide, a vector having said nucleic acid molecule, a recombinant virus particle having said vector and a capsid protein, a cell having said vector, a cell transformed with said vector, or a combination thereof.

[0333] In some embodiments, the present disclosure provides a method for treating a disease caused by damage to ribbon synapses in the inner ear in a subject in need, comprising administering to the subject a composition comprising a polypeptide having an amino acid sequence of a TAFA protein or a fragment thereof or a variant thereof, a nucleic acid molecule encoding said polypeptide, a vector having said nucleic acid molecule, a recombinant virus particle having said vector and a capsid protein, a cell having said vector, a cell transformed with said vector, or a combination thereof.

[0334] In some embodiments, the present disclosure provides a method for inducing ribbon synapse formation in the inner ear of a subject in need, comprising administering to the subject a composition comprising a polypeptide having an amino acid sequence of a TAFA protein or a fragment thereof or a variant thereof, a nucleic acid molecule encoding said polypeptide, a vector having said nucleic acid molecule, a recombinant virus particle having said vector and a capsid protein, a cell having said vector, a cell transformed with said vector, or a combination thereof.

[0335] In some embodiments, the present disclosure provides a method for improving the function of ribbon synapses in the inner ear of a subject in need, comprising administering to the subject a composition comprising a polypeptide having an amino acid sequence of a TAFA protein or a fragment thereof or a variant thereof, a nucleic acid molecule encoding said polypeptide, a vector having said nucleic acid molecule, a recombinant virus particle having said vector and a capsid protein, a cell having said vector, a cell transformed with said vector, or a combination thereof.

[0336] In some embodiments, the present disclosure provides a method for increasing the number of ribbon synapses in the inner ear of a subject in need, comprising administering to the subject a composition comprising a polypeptide having an amino acid sequence of a TAFA protein or a fragment thereof or a variant thereof, a nucleic acid molecule encoding said polypeptide, a vector having said nucleic acid molecule, a recombinant virus particle having said vector and a capsid protein, a cell having said vector, a cell transformed with said vector, or a combination thereof.

[0337] In some embodiments, the present disclosure provides a method for inhibiting damage to spiral ganglion neurons in a required target, comprising administering to the target a composition comprising a polypeptide having an amino acid sequence of a TAFA protein or a fragment thereof or a variant thereof, a nucleic acid molecule encoding said polypeptide, a vector having said nucleic acid molecule, a recombinant virus particle having said vector and a capsid protein, a cell having said vector, a cell transformed with said vector, or a combination thereof.

[0338] In some embodiments, the present disclosure provides a method for growing a neurite of a spiral ganglion neuron in a subject in need, comprising administering to the subject a composition comprising a polypeptide having an amino acid sequence of a TAFA protein or a fragment thereof or a variant thereof, a nucleic acid molecule encoding said polypeptide, a vector having said nucleic acid molecule, a recombinant virus particle having said vector and a capsid protein, a cell having said vector, a cell transformed with said vector, or a combination thereof.

[0339] In some embodiments, the present disclosure provides a method for increasing the branching points of a spiral ganglion in a required target, comprising administering to the target a composition comprising a polypeptide having an amino acid sequence of a TAFA protein or a fragment thereof or a variant thereof, a nucleic acid molecule encoding said polypeptide, a vector having said nucleic acid molecule, a recombinant virus particle having said vector and a capsid protein, a cell having said vector, a cell transformed with said vector, or a combination thereof.

[0340] By using the virus delivery system described herein (such as a vector containing the nucleic acid molecule or a recombinant virus particle containing the vector and a capsid protein), the composition described herein 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, using the virus delivery system described herein drastically reduces the number of administrations of the composition, thereby enabling a doctor, patient, or subject to avoid the inconvenience caused by repeated administration of the composition. Depending on the patient's symptoms or needs, the composition may be administered at least 2 to 3 times initially at intervals of 1 to 2 weeks, and then once every 2 to 3 months, every 6 months, every 1 year or more, or every 2 to 10 years or more.

[0341]

[0342] The features and advantages of the present invention are summarized as follows:

[0343] (i) The present invention provides a polypeptide (e.g., a polypeptide comprising an imanosan sequence of a TAFA protein, a TAFA protein fragment, or a variant thereof) having the ability to induce or increase the number of ribbon synapses in the inner ear, improve the function of ribbon synapses, or increase the growth of neurites or branching points of spiral ganglia.

[0344] (ii) Additionally, the present invention provides a therapeutic use of a pharmaceutical composition comprising the polypeptide, a nucleic acid molecule encoding the polypeptide, a vector comprising the nucleic acid molecule, a recombinant virus particle comprising the vector and a capsid protein, a cell comprising the vector, a cell transformed with the vector, or a combination thereof.

[0345] (iii) The composition of the present invention can be usefully used as a treatment for inner ear diseases by preventing or improving damage or abnormalities in the auditory system or balance system (vestibular system).

[0346]

[0347] Figure 1 shows the results of measuring CtBP2 spots in the outer and inner hair cells of the cochlea after administration of TAFA4 to mice (Figure 1a: photograph, Figure 1b: graph comparing counts) (***p< 0.001).

[0348] Figure 2 shows the results of measuring CtBP2 spots in the outer and inner hair cells of the cochlea after administering the TAFA family of TAFA1, TAFA2, TAFA3, TAFA4, and TAFA5 to mice (*p< 0.05, **p< 0.01,***p< 0.001).

[0349] Figure 3a shows the results of analyzing changes in neurite length caused by full-length TAFA4 protein in the MED17.11 cell line, and Figure 3b shows the results of analyzing changes in branch points caused by full-length TAFA4 protein in the MED17.11 cell line. * indicates a p-value <0.05.

[0350] Figure 4a shows the results of analyzing changes in neurite length caused by TAFA4 peptide fragments (TAFA4 fragments 1-3, F1-3) in the MED17.11 cell line, and Figure 4b shows the results of analyzing changes in branch points caused by TAFA4 peptide fragments (F1-F3) in the MED17.11 cell line.

[0351] Figure 5 shows the sequence of peptides of TAFA4 (TAFA protein and fragments thereof (F1-F8)) according to an embodiment of the present invention.

[0352] Figure 6a shows the results of analyzing changes in neurite length caused by TAFA4 peptide fragments (TAFA4 fragments 4-8, F4-F8) in the MED17.11 cell line, and Figure 6b shows the results of analyzing changes in branch points caused by TAFA4 peptide fragments (F4-F8) in the MED17.11 cell line. ** indicates a p-value <0.01.

[0353] Figure 7 shows the results of confirming changes in auditory brainstem response thresholds in the control group (G1), noise-induced hearing loss group (G2), and TAFA4 overexpressing AAV administration group (G3).

[0354] Figure 8 shows the results of confirming changes in modulated otoacoustic emission thresholds in the control group (G1), the noise-induced hearing loss group (G2), and the TAFA4 overexpressing AAV administration group (G3).

[0355]

[0356] The present invention will be described in more detail below through embodiments. These embodiments are intended solely to explain the invention more specifically, and it will be obvious to those skilled in the art that the scope of the invention is not limited by these embodiments according to the gist of the invention.

[0357]

[0358] Examples

[0359] [Example 1] Evaluation of the protective efficacy of TAFA4 protein against synaptic damage

[0360] In the auditory system, hair cells and spiral ganglion neurons cooperate in a complex manner to enable hearing. Hair cells located in the cochlea of ​​the inner ear act as mechanoreceptors that convert incoming sound waves into electrical signals. A crucial connection in this process exists at the synapse between the hair cells and the spiral ganglion neurons. At this synapse, neurotransmitters are released from the hair cells in response to mechanical stimuli, facilitating the transmission of electrical signals to the spiral ganglion neurons.

[0361] These synapses are critical because their destruction or damage due to aging, noise exposure, ototoxic drugs, or genetic mutations can lead to sensorineural hearing loss. In such cases, impaired transmission of auditory signals due to synapses mediating hair cell-spiral ganglion neurons within the auditory system—also known as ribbon synapses—ultimately impairs hearing sensitivity and hearing. Furthermore, damage to ribbon synapses in the vestibular system causes balance system problems such as chronic vestibular decline, age-related vestibular decline, and auditory neuropathy. Understanding the complexity of these synaptic connections is essential for the mechanisms and potential treatments of inner ear diseases, including hearing loss and vestibular disorders.

[0362] In addition, CtBP2 is a key factor in maintaining the structural and functional integrity of ribbon synapses present in the auditory and vestibular systems, and the degree of damage, protection, and / or creation of ribbon synapses can be histologically evaluated by quantifying it.

[0363] The therapeutic efficacy of the TAFA protein provided by the present invention for hearing loss was evaluated in the organ of Corti of mice (4 days old, Nara Biotech). Overexpression of TAFA4 was induced by infecting the organ of Corti, located in the cochlea, with AAV, and changes in CtBP2, a ribbon synapse marker involved in signal transmission between hair cells and spiral ganglion neurons, were observed. The amino acid sequence of TAFA4 is shown in Table 1.

[0364] Amino acid sequence of recombinant human TAFA4 peptide Peptide Name Length (aa) Amino Acid Sequence (Sequence, N'→C') Recombinant human TAFA4 (Sequence No. 86) 105SQHLRGHAGH HQIKQGTCEV VAVHRCCNKN RIEERSQTVK CSCFPGQVAG TTRAQPSCVE ASIVIQKWWC HMNPCLEGED CKVLPDYSGW SCSSGNKVKT TKVTR

[0365]

[0366] 1.1. Resection of the Trachea of ​​Corti

[0367] Poly-D-Lysine (Sigma-Aldrich) was dispensed onto an 8-well chamber slide glass (SPL) for culturing organelles of Corti, and the slide glass was treated in a 37°C cell incubator (5% CO2) for 6 hours. After removing the supernatant, Laminin (Gibco) was dispensed and the slide glass was treated overnight in a 37°C cell incubator (5% CO2) to prepare the slide glass for culturing organelles of Corti.

[0368] The following day, the organ of Corti was excised from mice and cultured on prepared slide glasses. To obtain the organ of Corti, the heads of 4-day-old mice were first decapitated and the scalp removed. The sagittal and coronal sutures were then resected to separate the left and right hemispheres. The separated hemispheres were immersed in cold HBSS (Gibco) to minimize damage to the organ of Corti. The brains were removed from each hemisphere to expose the cochlea, and the otosacs were resected. Subsequently, the spiral ligament and Reisner's membrane were removed, and the organ of Corti was trimmed by dividing it into three sections and removing the tectorial membrane. The organ of Corti was attached to the prepared slide glass with the hair cells facing upward. High glucose DMEM (Gibco) containing 1% N2 supplement (Gibco) and 10 μg / mL Ampicillin (Merck) was used as the culture medium. The culture medium was added, and the organ was stabilized overnight in a 37°C incubator (5% CO2). The next day, all of the existing culture medium was removed and replaced with a culture medium containing 10% fetal bovine serum (Gibco). Subsequently, AAV9 inserted with eGFP and TAFA4 overexpression vectors was cultured at a rate of 5x10 10 The cells were treated with viral genome (vg) / mL and cultured for 3 days in a cell culture incubator (5% CO2) at 37℃.

[0369]

[0370] 1.2. Immunostaining

[0371] After removing all of the culture medium containing AAV9, the specimens were fixed in 4% paraformaldehyde (HanLAB) at 4°C for 20 minutes. Subsequently, the remaining fixative was removed by washing three times for 10 minutes each with 1X PBS (Gibco). After washing, a blocking solution containing 3% bovine serum albumin (Sigma) and 0.1% Triton X-100 (Sigma) in 1X PBS was added, and the specimens were incubated at room temperature for 1 hour to block non-specific binding. Afterward, the primary antibody, anti-mouse CtBP2 (BD Bioscience), was added, and the specimens were incubated overnight at 4°C. The next day, the specimens were washed three times with PBS, treated with Hoechst and fluorescently labeled secondary antibodies, and incubated at room temperature for 1 hour. After incubation, the specimens were washed three times for 10 minutes each, and the organs of Corti were photographed at 40X using a confocal microscope (Leica). In the captured images, the number of CtBP2 spots appearing in outer hair cells and inner hair cells was counted and compared using the ImageJ program. Statistical significance was confirmed using the unpaired Student's test.

[0372] It was confirmed that the CtBP2 spots appearing in outer and inner hair cells were statistically significantly increased in the group overexpressing TAFA4 compared to the group overexpressing eGFP (***p< 0.001) (Figs. 1a and 1b). These results highlight that TAFA4 described herein has protective properties against synapse loss induced by damage to hair cells that occurs during the separation process of mouse cochlear ex vivo grafts.

[0373]

[0374] [Example 2] Evaluation of the synapse formation-promoting efficacy of the TAFA protein group

[0375] The TAFA protein family includes TAFA1, TAFA2, TAFA3, and TAFA5 in addition to TAFA4, and they exhibit high sequence identity characterized by common repetitive cysteine ​​structures (Sarver DC et al., "An Emerging Family of Neurokines with Diverse Functions in the Central and Peripheral Nervous System", ACS Chem Neurosci., 17;12(6):945-958, (Mar, 2021)). Based on this, we further investigated whether TAFA1, TAFA2, TAFA3, and TAFA5, which belong to the TAFA protein family as well as TAFA4, could increase the number of ribbon synapses damaged by organocorti excision. Organocorti was excised using the same method as in Example 1, and 5x10 AAV8 vectors inserted with eGFP and TAFA 1 through 5 overexpression vectors were... 10 The cells were treated with vg / mL and cultured for 5 days in a 37°C cell incubator (5% CO2). Subsequently, immunostaining was performed in the same manner as in Example 1, and synapses were observed by counting CtBP2 spots within the hair cells. Statistical significance was confirmed using one-way ANOVA (Dunnett's post-hoc test).

[0376] When the number of CtBP2 spots was counted and compared, it was confirmed that the number of CtBP2 spots increased statistically significantly in the outer and inner hair cells of the groups overexpressing TAFA1, TAFA2, TAFA3, and TAFA4 (*p< 0.05, **p< 0.01, ***p< 0.001). On the other hand, in the case of TAFA5, the number of CtBP2 spots decreased (Fig. 2). These results suggest that, despite being in the same family, TAFA5 causes more damage to ribbon synapses, whereas TAFA4 and its family, TAFA1 to 3, described herein possess the ability to maintain and generate ribbon synapses between hair cells and spiral ganglion neurons, and that the sequence identity of their common sequence plays an important role in the protection and generation of ribbon synapses.

[0377] The sequence identity between the common sequences of TAFA1 to TAFA4 above is as shown in Table 2.

[0378] TAFA1, TAFA2, TAFA3, and TAFA4 are identical sequences of each species (93 aa). scrofa919075.8%TAFA1RabbitOryctolagus cuniculus919175.8%TAFA1RatRattus norvegicus919275.8%TAFA1MouseMus musculus919375.8%TAFA1ChickenGallus gallus919475.8%TAFA1Komodo dragonVaranus komodoensis919576.9%TAFA1Wall lizardPodarcis muralis919676.9%TAFA1Fence lizardSceloporus undulatus919776.9%TAFA1FrogXenopus tropicalis_isoform1929876.1%TAFA1FrogXenopus tropicalis_isoform2929976.1%TAFA1FishDanio rerio_isoform19310074.2%TAFA1FishDanio rerio_isoform29310175.3%TAFA2HumanHomo sapiens9310286.0%TAFA2MonkeyMacaca fascicularis9310386.0%TAFA2PigSus scrofa9310486.0%TAFA2RabbitOryctolagus cuniculus9310586.0%TAFA2RatRattus norvegicus_isoform19310682.8%TAFA2RatRattus norvegicus_isoform29310784.9%TAFA2MouseMus musculus9310884.9%TAFA2ChickenGallus gallus_isoform19310983.9%TAFA2ChickenGallus gallus_isoform29311087.1%TAFA2Komodo dragonVaranus komodoensis9311184.9%TAFA2Wall lizardPodarcis muralis_isoform19111285.7%TAFA2Wall lizardPodarcis muralis_isoform29311387.1%TAFA2Fence lizardSceloporus undulatus9311487.1%TAFA2GeckoGekko japonicus9211583.7%TAFA2FrogXenopus tropicalis9311684.9%TAFA2FishDanio rerio9311782.8%TAFA3HumanHomo sapiens9311882.8%TAFA3MonkeyMacaca fascicularis9311982.8%TAFA3PigSus scrofa9312084.9%TAFA3RabbitOryctolagus cuniculus9312182.8%TAFA3RatRattus norvegicus9312282.8%TAFA3MouseMus musculus9312383.9%TAFA3ChickenGallus gallus9312484.9%TAFA3Komodo dragonVaranus komodoensis9312586.0%TAFA3Wall lizardPodarcis muralis9312683.9%TAFA3Fencee lizardSceloporus undulatus9312786.0%TAFA3FrogXenopus tropicalis9312887.1%TAFA3FishDanio rerio9312981.7%TAFA4MonkeyMacaca fascicularis93130100.0%TAFA4PigSus scrofa93131100.0%TAFA4RabbitOryctolagus cuniculus9313296.8%TAFA4RatRattus norvegicus9313396.8%TAFA4MouseMus musculus9313495.7%TAFA4ChickenGallus gallus9313596.8%TAFA4Komodo dragonVaranus komodoensis9313694.6%TAFA4Wall lizardPodarcis muralis9313796.8%TAFA4Fence lizardSceloporus undulatus9313895.7%TAFA4GeckoGekko japonicus9313987.1%TAFA4FrogXenopus tropicalis9314092.5%TAFA4FishDanio rerio9314187.1%.

[0379] Sequence identity was confirmed using Clustal Omega.

[0380]

[0381] [Example 3] Evaluation of the efficacy of TAFA protein in neurite growth

[0382] Spiral ganglion neurons (SGNs) are a key component of the auditory system. These specialized neurons serve as major conduits that transmit electrical impulses generated by hair cells in the cochlea to the central auditory pathways in the brain. Their role involves the encoding and transmission of important auditory parameters, including sound frequency, intensity, and temporal characteristics. However, sensorineural hearing loss, the most common form of hearing impairment, is accompanied by damage to these neurons, and enhancing neurite outgrowth is useful for the treatment of such inner ear disorders (Kempfle et al., "A Novel Small Molecule Neurotrophin-3 Analogue Promotes Inner Ear Neurite Outgrowth and Synaptogenesis In vitro", Front. Cell. Neurosci., 15 (July, 2021)).

[0383] In order to evaluate the regenerative capacity of SGN under the aforementioned inner ear disease, we evaluated whether the TAFA proteins described herein possess the ability to increase neuronal neurite growth and branching points. Mouse MED17.11 cell lines were differentiated into ganglion neurons, and changes in neurite length or branching points induced by recombinant human TAFA4 (hereinafter rhTAFA4) were evaluated.

[0384] MED17.11 cell lines were cultured in a 33°C incubator (5% CO2), and undifferentiated cells were cultured using DMEM / F-12 culture medium (Gibco) supplemented with 10% fetal bovine serum (Gibco), 1% penicillin / streptomycin (Gibco), 5 ng / mL interferon-gamma (R&D systems), and 0.5% chicken embryonic extract (Sera lab). The method for evaluating the efficacy of TAFA4 is as follows. Undifferentiated MED17.11 cells were harvested using 0.25% trypsin-EDTA (Gibco). After centrifuging the harvested cells and removing the supernatant, the cell pellet was resuspended in differentiation induction medium. The differentiation induction medium used was DMEM / F-12 culture medium supplemented with 10% fetal bovine serum (Gibco), 1% penicillin / streptomycin (Gibco), 10 ng / mL Fibroblast growth factor 2 (R&D Systems), 0.5 mM dibutyryl cAMP (Sigma), 25 μM Forskolin (Sigma), 5 μg / mL Y-27632 (Chemdea), 100 ng / mL beta Nerve growth factor (R&D Systems), and 10 ng / mL Glial-derived neurotrophic factor (R&D Systems). MED17.11 cells resuspended in the differentiation induction medium were cultured in 96-well cell culture plates (Thermofisher) with 1 x 10 wells per well. 4100 μL of cells / mL was dispensed. Subsequently, 1 μM of rhTAFA4 (R&D systems, Table 1) was added to the experimental group, and an equal volume of Phosphate buffered saline (Gibco) was added to the control group, after which the cells were incubated in a 37°C incubator (5% CO2). During incubation, images of the cells were taken using Incucyte (Satorius), and the neurite length and branch points in the MED17.11 cells were analyzed using the Incucyte software. A total of three replicate experiments were conducted, and statistical significance was confirmed using the Student's St-test.

[0385] After 4 days, it was confirmed that the neurite length and branch point of MED17.11 cells increased statistically significantly in the group treated with rhTAFA4 compared to the control group (Figs. 3a and 3b).

[0386]

[0387] [Example 4] Evaluation of the efficacy of TAFA protein fragment in neurite growth

[0388] 4.1 Evaluation of the efficacy of fragments 1 to 3

[0389] Based on the results of Example 3 above, the inventors synthesized three TAFA4 peptide fragments (TAFA4 fragments 1-3, F1-F3) containing regions exhibiting high sequence identity as shown in Table 3 to identify fragments exhibiting biological efficacy of TAFA4. The peptide synthesis was commissioned to Abclon.

[0390] Amino acid sequence of TAFA4 peptide fragments 1–3 Peptide Name Length (aa) Amino acid sequence (Sequence, N'→C') TAFA4 Fragment 1 (Sequence No. 148) 35HQIKQGTCEV VAVHRCCNKN RIEERSQTVK CSCFP TAFA4 Fragment 2 (Sequence No. 75) 31GQVAGTTRAQ PSCVEASIVI QKWWCHMNPC L TAFA4 Fragment 3 (Sequence No. 52) 29EGEDCKVLPD YSGWSCSSGN KVKTTKVTR

[0391] MED17.11 cells resuspended in differentiation induction medium in the same manner as in Example 3 were placed in 1 x 10 wells of a 96-well cell culture plate. 4 After dispensing 100 μL of cells / mL, 5 μM of TAFA4 peptide fragments 1-3 and an equal volume of dimethyl-sulfoxide (DMSO, Sigma) were added to the control group, and the cells were incubated in a 37°C incubator (5% CO2). After 4 days of incubation, neurite length and branch points were analyzed using Incucytes. A total of three replicate experiments were performed, and an increasing trend in both neurite length and branch points was observed in the groups treated with TAFA4 fragment 2 (F2) and TAFA4 fragment 3 (F3) (Figures 4a and 4b).

[0392] The species sequences of TAFA4 fragment 2 (F2) and fragment 3 (F3) are as shown in Tables 4 and 5 below.

[0393] Amino acid sequences by species of TAFA4 fragment 2 Species (Scientific Name) SEQ ID NOSEQUENCEA.A Human (Homo sapiens), Monkey (Macaca fascicularis), Pig (Sus scrofa) Sequence No. 75 GQ VAGTTRAQ PSCVEASIVIQKWWCHMNPCL31 Rabbit (Oryctolagus cuniculus) Sequence No. 76 GQ VAGTTQAQ PSCVEASIVAQKWWCHMNPCL31 Rat (Rattus norvegicus) Sequence No. 77 GQ VAGTTRAQ PSCVEASIVIEKWWCHMDPCL31 Mouse (Mus musculus) Sequence No. 78 GQ VAGTTRAQ PSCVEAAIVIEKWWCHMNPCL31 Chicken (Gallus gallus) Sequence No. 79 GQ VAGTTRAQ PSCVEASIVLQKWWCHMNPCL31 Komodo dragon (Varanus komodoensis) Sequence No. 80GQVAGTTRAQPSCVEAAIVVQKWWCHMNPCL31 Wall lizard (Podarcis muralis) Sequence No. 81GQVAGTTRAQPSCVEAAIVIQKWWCHMNPCL31 Fence lizard (Sceloporus undulatus) Sequence No. 82GQVAGTTRSQPSCVEAAIVIQKWWCHMNPCL31 Gecko (Gekko japonicus) Sequence No. 83GQVAGTTRTQPSCVEAAIVIQKWWCQMSPCL31 Frog (Xenopus tropicalis) Sequence No. 84GQVAGTTRAQPSCVEASIVIQKWWCHMNPCM31 Fish (Danio rerio) Sequence No. 85GQVAGTTRAQPSCVEASIVLQKWWCQMHPCL31

[0394] The above fragment 2 and the sequence reflecting the interspecies variation thereof are the same as sequence number 147.

[0395] Amino acid sequence by species of TAFA4 fragment 3 (scientific name) SEQ ID NOSEQUENCEA.AHuman (Homo sapiens), Monkey (Macaca fascicularis), Pig (Sus scrofa), Rabbit (Oryctolagus cuniculus) SEQ ID NO. 52EGEDCKVLPDYSGWSCSSGNKVKTTKVTR29Rat (Rattus norvegicus), Mouse (Mus) musculus) SEQ ID NO: 53EGEDCKVLPDSSGWSCSSGNKVKTTKVTR29Chicken (Gallus gallus), Wall lizard (Podarcis muralis)SEQ ID NO: 54DGEDCKVLPDYSGWSCSSGNKVKTTKVTR29Komodo dragon (Varanus komodoensis), Fence lizard (Sceloporus undulatus)SEQ ID NO: 55EGEECKVLPDYSGWSCSSGNKVKTTKVTR29Gecko (Gekko japonicus) Sequence No. 56EGEECKVLPDSSGWSCSSGNKVKTTKVTR29 Frog (Xenopus tropicalis) Sequence No. 57EGEECKVLPDLTGWSCSSGNKIKTTKVTR29 Fish (Danio rerio) Sequence No. 58DGEECKALPDLTGWSCSTGNKVKTTKVTR29

[0396] The above fragment 3 and the sequence reflecting the interspecies variation thereof are as in sequence number 144.

[0397]

[0398] 4.2 Evaluation of the Efficacy of Detailed Fragments

[0399] Based on Example 4.1, in which TAFA fragments 2 (F2) and 3 (F3) were effective, more subdivided TAFA4 peptide fragments (TAFA4 fragments 4-8, F4-F8) were synthesized as shown in Table 6 to identify the key sites exhibiting biological efficacy. The peptide synthesis was commissioned to Abclon. The location of each fragment is shown in Fig. 5.

[0400]

[0401] Amino acid sequence of TAFA4 peptide fragments 4-8 Peptide Name Length (aa) Amino acid sequence (Sequence, N'→C') TAFA4 Fragment 4 (Sequence No. 149) 20HQIKQGTCEV VAVHRCCNKNTAFA4 Fragment 5 (Sequence No. 59) 32RIEERSQTVK CSCFPGQVAG TTRAQPSCVE STAFA4 Fragment 6 (Sequence No. 150) 17FPGQVAGTTR AQPSCVETAFA4 Fragment 7 (Sequence No. 28) 22IVIQKWWCHM NPCLEGEDCK VLTAFA4 Fragment 8 (Sequence No. 151) 21PDYSGWSCSS GNKVKTTKVT R

[0402] In the same manner as in Example 4.1, 5 μM of TAFA4 peptide fragment and an equal volume of DMSO were added, and the cells were incubated in a 37°C incubator (5% CO2). After culturing for 4 days, cell images were taken using Incucyte (Satorius), and neurite length and branch points were analyzed. Two replicate experiments were performed, and statistical significance was confirmed via one-way ANOVA (Dunnett's post-hoc test). The neurite length showed an increasing trend in TAFA4 fragment 5 (F5) and fragment 7 (F7) (Fig. 6a). Similarly, an increasing trend in branch points was observed only in TAFA4 fragment 5 (F5) and fragment 7 (F7) (Fig. 6b).

[0403]

[0404] The interspecies sequences of TAFA4 fragment 5 (F5) and fragment 7 (F7) are as shown in Tables 7 and 8 below.

[0405]

[0406] Comparison of Interspecies Homology of TAFA4 Peptide Fragment 5 Species (Scientific Name) SEQ ID NO Amino Acid Sequence (Sequence, N'→C') Sequence Identity Human (Homo sapiens), Monkey (Macaca fascicularis), Pig (Sus scrofa), Rat (Rattus norvegicus), Chicken (Gallus gallus), Frog (Xenopus tropicalis) Sequence No. 59 RIEERS QTVK CSCFPG QVAG TTRA QPS CVE AS 100.0% Rabbit (Oryctolagus cuniculus) Sequence No. 60 RIEERS QTVK CSCFPG QVAG TTQA QPS CVE AS 96.9% Mouse (Mus musculus), Wall lizard (Podarcis muralis) Sequence No. 61 RIEERS QTVK CSCFPG QVAG TTRA QPS CVE AA 96.9% Komodo dragon (Varanus komodoensis) Sequence No. 62 RIEERSQTVK CSCLPGQVAG TTRAQPSCVE AA93.8% Fence lizard (Sceloporus undulatus) Sequence No. 63 RIEERSQTVK CSCFPGQVAG TTRSQPSCVE AA93.8% Gecko (Gekko japonicus) Sequence No. 64 RIELSQTGN CSCLPGQVAG TTRTQPSCVE AA81.3% Fish (Danio rerio) Sequence No. 65 KIEERSQTVK CSCFPGQVAG TTRAQPSCVE AS96.9%

[0407]

[0408] Comparison of interspecies homology of TAFA4 peptide fragment 7Species (Scientific Name) SEQ ID NO Amino Acid Sequence (Sequence, N'→C') Sequence Identity Human (Homo sapiens), Monkey (Macaca fascicularis), Pig (Sus scrofa) Sequence No. 28 IVIQKWWCHM NPCLEGEDCK VL 100.0% Rabbit (Oryctolagus cuniculus) Sequence No. 29 IVAQKWWCHM NPCLEGEDCK VL 95.5% Rat (Rattus norvegicus) Sequence No. 30 IVIEKWWCHM DPCLEGEDCK VL 90.9% Mouse (Mus musculus) Sequence No. 31 IVIEKWWCHM NPCLEGEDCK VL 95.5% Chicken (Gallus gallus) Sequence No. 32 IVLQKWWCHM NPCLDGEDCK VL 90.9% Komodo dragon (Varanus komodoensis) Sequence No. 33 IVVQKWWCHM NPCLEGEECK VL90.9% Wall lizard (Podarcis muralis) Sequence No. 34 IVIQKWWCHM NPCLDGEDCK VL95.5% Fence lizard (Sceloporus undulatus) Sequence No. 35 IVIQKWWCHM NPCLEGEECK VL95.5% Gecko (Gekko japonicus) Sequence No. 36 IVIQKWWCQM SPCLEGEECK VL86.4% Frog (Xenopus tropicalis) (Sequence No. 32) Sequence No. 37 IVIQKWWCHM NPCMEGEECK VL90.9% Fish (Danio rerio) Sequence No. 38 IVLQKWWCQM HPCLDGEECK AL72.7%

[0409] Fragments 2 and 5, which exhibited the above efficacy, share the GQVAGTTRAQPSCVEAS sequence (17 amino acids, referred to as fragment 2.5), and fragments 3 and 7 share the EGEDCKVL sequence (8 amino acids, referred to as fragment 3.7). Therefore, the effect of increasing the length and branching points of the neurites in Examples 3 and 4 is determined to be due to the polypeptide containing the sequence of fragment 2.5 or the sequence of fragment 3.7, which is the common sequence of the fragments.

[0410] As a result of comparing the sequence of the above TAFA4 fragment 2.5 with the sequences of TAFA 1 to 3 interspecies variants, it was confirmed that there is only a difference of about 1 to 4 amino acid sequences from the human TAFA4 fragment 2.5, thereby confirming that the homology is very high.

[0411] In addition, by comparing the sequence of the above TAFA4 fragment 3.7 with the sequences of TAFA 1 to 3 interspecies variants, it was confirmed that the human TAFA4 fragment 3.7 differed from the human fragment only by about 1 to 3 amino acids, thereby confirming that fragment 3.7 also has very high homology.

[0412] The results of comparing the sequence of the above human TAFA4 fragment with the sequences of interspecies variants of TAFA 1 to 4 are as shown in Tables 9 (Fragment 2.5) and 10 (Fragment 3.7) below.

[0413] Sequences of the TAFA protein fragment 2.5 exhibiting effect and its interspecies variantsTAFA Species SEQ ID NOSequence, N'→C'TAFA4HumanMonkeyPigRatChickenFrogFish Sequence No. 15GQVAG TTRAQPSCVE ASRabbit Sequence No. 16GQVAG TTQAQPSCVE ASMouseKomodo dragonWall lizard Sequence No. 17GQVAG TTRAQPSCVE AAFence lizard Sequence No. 18GQVAG TTRSQPSCVE AAGecko Sequence No. 19GQVAG TTRTQPSCVE AATAFA1HumanMonkeyPigRabbitRatMouseChickenKomodo dragonWall lizardFence lizardFrog Sequence No. 20GKVAG TTRNRPSCVDASFish Sequence No. 21GKVAG TTRNKPSCVDASGecko Sequence No. 22GKVAG TTRNRPSCVD--TAFA2HumanMonkeyPigRabbitRatMouseChickenKomodo dragonWall lizardFence lizardGeckoFish Sequence No. 23GQVAG TTRAAPSCVDASFrog Sequence No. 24GQVAG TTRATPSCVDASTAFA3HumanMonkeyPigRabbitRatMouse Sequence No. 25GQVAG TTRAKPSCVDASChickenKomodo dragonFence lizardFrogFish Sequence No. 26GQVAG TTRAAPSCVDASWall lizard Sequence No. 27GQVAG TTHAAPSCVDAS

[0414] Differing amino acids are indicated in bold. The sequence of fragment 2.5 and its interspecies variation is the same as SEQ ID NO. 145.

[0415]

[0416] Sequences of TAFA protein fragment 3.7 exhibiting effect and its interspecies variantsTAFA Species SEQ ID NOSequence, N'→C'TAFA4HumanMonkeyPigRabbitRatMouse Sequence No. 1EGEDCK VLChickenWall lizard Sequence No. 2DGEDCK VLKomodo dragonFence lizardGeckoFrog Sequence No. 3EGEECK VLFish Sequence No. 4DGEECKALTAFA1HumanMonkeyPigRabbitRatMouseChickenKomodo dragonWall lizardFence lizardFrogFish Sequence No. 5EGEECKTLTAFA2HumanMonkeyPigRabbitRatMouseChickenKomodo dragonWall lizardFence lizardGecko Sequence No. 6EGEECK VLFrog Sequence No. 7EGEECKILFish Sequence No. 8DGEECK VLTAFA3HumanMonkeyPigRabbit Sequence No. 9PGEECK VLRatMouse Sequence No. 10LGEECK VLChicken Sequence No. 11 AGEECK VLKomodo dragonWall lizardFence lizard Sequence No. 12 EGEECK VLFrog Sequence No. 13 EGEDCK VLFish Sequence No. 14 DGEECK VL

[0417] Differing amino acids are indicated in bold. The sequence of fragment 3.7 and its interspecies variation is the same as SEQ ID NO. 142.

[0418]

[0419] [Example 5] Comparison of Homology between TAFA4 Peptide Fragment and TAFA 1 to 3 Peptide Fragments

[0420] In Example 4, it was confirmed that the interspecies homology of the TAFA4 fragment was 70% or higher (F5: 81.3% or higher, F7: 72.7% or higher). It is known that there is high sequence identity between TAFA4 and the TAFA protein group. Therefore, it was hypothesized that the common sequence of various TAFA peptide fragments would have an effect on increasing neurite length and branch points. Sequence homology (Tables 11 to 16) was compared between the TAFA1, TAFA2, and TAFA3 peptide fragments of vertebrate mammals (Human, Monkey, Pig, Rabbit, Rat, Mouse), birds (Chicken), reptiles (Komodo dragon, Wall lizard, Fence lizard, Gecko), amphibians (Frog), and fish (Fish) and human TAFA4 fragment 5 (F5) and fragment 7 (F7), and the consensus sequence was identified.

[0421]

[0422] Comparison of Homology Between Human TAFA4 Fragment 5 and TAFA1 Peptide Fragment 5Species (scientific name) SEQ ID NO Amino acid sequence (Sequence, N'→C') Sequence identity Human (Homo sapiens), Monkey (Macaca fascicularis), Pig (Sus scrofa), Rabbit (Oryctolagus cuniculus), Rat (Rattus norvegicus), Mouse (Mus musculus), Chicken (Gallus gallus), Komodo dragon (Varanus komodoensis), Wall lizard (Podarcis muralis), Fence lizard (Sceloporus undulatus), Frog (Xenopus tropicalis) SEQ ID NO 66 RIEERSQTVK CSCLPGKVAG TTRNRPSCVD AS84.4% Gecko (Gekko japonicus) SEQ ID NO 67 RIEERSQTVK CSCLPGKVAG TTRNRPSCVD --78.1% Fish (Danio rerio) SEQ ID NO 68 KIEERSQTVK CSCLPGKVAG TTRNRPSCVD AS81.3%

[0423]

[0424] Comparison of Homology Between Human TAFA4 Fragment 5 and TAFA2 Peptide Fragment 5Species (scientific name) SEQ ID NO Amino acid sequence (Sequence, N'→C') Sequence identity Human (Homo sapiens), Monkey (Macaca fascicularis), Pig (Sus scrofa), Rabbit (Oryctolagus cuniculus), Rat (Rattus norvegicus), Mouse (Mus musculus), Chicken (Gallus gallus), Komodo dragon (Varanus komodoensis), Wall lizard (Podarcis muralis), Fence lizard (Sceloporus undulatus), Gecko (Gekko japonicus), Fish (Danio rerio) SEQ ID NO 69 KIEERSQTVK CSCFPGQVAG TTRAAPSCVD AS90.6% Frog (Xenopus tropicalis) SEQ ID NO 70 KIEERSQTVK CSCFPGQVAG TTRATPSCVD AS90.6%

[0425]

[0426] Comparison of Homology Between Human TAFA4 Fragment 5 and TAFA3 Peptide Fragment 5Species (Scientific Name) SEQ ID NO Amino Acid Sequence (Sequence, N'→C') Sequence Identity Human (Homo sapiens), Monkey (Macaca fascicularis), Pig (Sus scrofa) Sequence No. 71 RIEERS QTVK CSCFSG QVAG TTRAK PSCVD AS 90.6% Rabbit (Oryctolagus cuniculus), Rat (Rattus norvegicus), Mouse (Mus musculus) Sequence No. 72 RIEERS QTVK CSCLSG QVAG TTRAK PSCVD AS 87.5% Chicken (Gallus gallus), Komodo dragon (Varanus komodoensis), Fencee lizard (Sceloporus undulatus), Frog (Xenopus tropicalis), Fish (Danio rerio) Sequence No. 73 KIEERS QTVK CSCFPG QVAG TTRAAP SCVD AS 90.6% Wall lizard (Podarcis muralis) SEQ ID NO: 74KIEERSQTVK CSCFPGQVAG TTHAAPSCVD AS87.5%

[0427] The above fragment 5 and the sequence reflecting the interspecies variation thereof are the same as sequence number 146.

[0428]

[0429] Comparison of Homology Between Human TAFA4 Fragment 7 and TAFA1 Peptide Fragment 7Species (scientific name) SEQ ID NO Amino acid sequence (Sequence, N'→C') Sequence identity Human (Homo sapiens), Monkey (Macaca fascicularis), Pig (Sus scrofa), Rabbit (Oryctolagus cuniculus), Rat (Rattus norvegicus), Mouse (Mus musculus), Chicken (Gallus gallus), Komodo dragon (Varanus komodoensis), Wall lizard (Podarcis muralis), Fence lizard (Sceloporus undulatus), Frog (Xenopus tropicalis), Fish (Danio rerio) Sequence number 39 IVIGKWWCEM EPCLEGEECK TL77.3%

[0430]

[0431] Comparison of Homology Between Human TAFA4 Fragment 7 and TAFA2 Peptide Fragment 7 Species (scientific name) SEQ ID NO Amino acid sequence (Sequence, N'→C') Sequence identity Human (Homo sapiens), Monkey (Macaca fascicularis), Pig (Sus scrofa), Rabbit (Oryctolagus cuniculus), Rat (Rattus norvegicus), Mouse (Mus musculus), Chicken (Gallus gallus), Komodo dragon (Varanus komodoensis), Wall lizard (Podarcis muralis), Fence lizard (Sceloporus undulatus) SEQ ID NO 40IVEQKWWCHM QPCLEGEECK VL86.4% Gecko (Gekko japonicus) SEQ ID NO 41IVEQKWWCQM QPCLEGEECK VL81.8% Frog (Xenopus tropicalis) SEQ ID NO 42IVEQKWWCHM QPCLEGEECK IL81.8% Fish (Danio rerio) SEQ ID NO 43IVAQKWWCQM QPCMDGEECK VL72.7%

[0432]

[0433] Comparison of Homology Between Human TAFA4 Fragment 7 and TAFA3 Peptide Fragment 7 Species (Scientific Name) Length (aa) Amino Acid Sequence (Sequence, N'→C') Sequence Identity Human (Homo sapiens), Monkey (Macaca fascicularis), Rabbit (Oryctolagus cuniculus) Sequence No. 44 IVLQRWWCQM EPCLPGEECK VL72.7% Pig (Sus scrofa) Sequence No. 45 IVLQKWWCQM EPCLPGEECK VL77.3% Rat (Rattus norvegicus), Mouse (Mus musculus) Sequence No. 46 IVLQKWWCQM EPCLLGEECK VL77.3% Chicken (Gallus gallus) Sequence No. 47 IVLQKWWCQM EPCLAGEECK VL77.3% Komodo dragon (Varanus komodoensis), Fencee lizard (Sceloporus undulatus) Sequence No. 48 IVLQKWWCQM QPCLEGEECK VL81.8%Wall lizard (Podarcis muralis) Sequence No. 49IVLQKWWCQM QPCVEGEECK VL77.3%Frog (Xenopus tropicalis) Sequence No. 50IVIQKWWCQM EPCLEGEDCK VL90.9%Fish (Danio rerio) Sequence No. 51IVAQKWWCQM QPCVDGEECK VL72.7%

[0434] The above fragment 7 and the sequence reflecting the interspecies variation thereof are the same as sequence number 143.

[0435] Additionally, the interspecies sequences of fragment 3 of TAFA1 to 3 (Table 17) and the interspecies sequence of fragment 2 (Table 18) are as follows:

[0436]

[0437] Interspecies sequence of fragment 3 of TAFA1 to 3TAFA Species SEQ ID NO Sequence, N'→ C'TAFA1 Human, Monkey, Pig, Rabbit, Rat, Mouse, Chicken, Frog(isoform2) Sequence No. 152 EGEECKTLPDNSGWMCATGNKIKTTR-IHTAFA1 Komodo dragon, Wall lizard, Fence lizard Sequence No. 153 EGEECKTLPDNSGWMCATGNKVKTTR-IHTAFA1 Frog(isoform1) Sequence No. 154 EGEECKTLPDNSGWMCATGNKIKTTR-FQTAFA1 Fish(isoform1) Sequence No. 155 EGEECKTLPDNSGWMCYSGNKIKTTRGNITAFA1 Fish(isoform2) Sequence No. 156 EGEECKTLPDNSGWMCYSGNKIKTTRNTHTAFA2 Human, Monkey, Pig, Rabbit, Rat(isoform2), Mouse Sequence No. 157 EGEECK VLPDRKGWS CSSGNK VKTTR VTHTAFA 2Rat (isoform 1), Chicken (form 1) Sequence No. 158 EGEECK VLPDRKGWS CSSGNK VKTTRAN VTAFA 2Chicken (isoform 2), Wall lizard (2), Fence lizard Sequence No. 159 EGEECK VLPDRKGWS CSSGNK VKTTR VTRTAFA 2Komodo dragon Sequence No. 160 EGEECK VLPDRKGWS CSSGNK VKTTR VSKTAFA 2Wall lizard sequence number 161EGEECKVLPDRKGWSCSSGNKVKTTRM--TAFA2Gecko sequence number 162EGEECKVLPDRKGWSCSSGNKVKTTRAN-TAFA2Frog sequence number 163EGEECKILPDQKGWSCASGNKVKTTKVTRTAFA2Fish sequence number 164DGEECKVLPDLKGWSCSTGNKVKTTKVTRTAFA3 Human, Monkey, Pig,Rabbit Sequence No. 165 PGEECK VLPDLSGWS CSSGHK VKTTK VTRTAFA3Rat Sequence No. 166 LGEECK VLPDLSGWSCSRGHK VKTTK VTRTAFA3Mouse Sequence No. 167 LGEECK VLPDLSGWS CSSGHK VKTTK VTRTAFA3Chicken Sequence No. 168 AGEECK VLPDLSGWS CSSGNK VKTTK VTRTAFA3Komodo dragon, Wall lizard, Fencee lizard Sequence No. 169 EGEECK VLPDLSGWS CSTGNK VKTTK VTRTAFA3Frog Sequence No. 170 EGEDCK VLPDLSGWS CSTGNK VKTTK VTRTAFA3Fish Sequence No. 171 DGEECK VLPDLTGWS CSTGNK VKTTK VTR,

[0438]

[0439] Interspecies sequence of fragment 2 of TAFA1 to 3TAFA Species SEQ ID NO Sequence, N'→ C'TAFA1 Human, Monkey, Pig, Rabbit, Rat, Mouse, Chicken, Komodo dragon, Wall lizard, Fence lizard, Frog Sequence No. 172 GK VAGTTRNRPSC VDASI VIGKWWCEMEPCLTAFA1 Fish Sequence No. 173 GK VAGTTRNKPSC VDASI VIGKWWCEMEPCLTAFA2 Human, Monkey, Pig, Rabbit, Rat, Mouse, Chicken, Komodo dragon, Wall lizard, Fence lizard Sequence No. 174GQVAGTTRAAPSCVDASIVEQKWWCHMQPCLTAFA2Gecko Sequence No. 175GQVAGTTRAAPSCVDASIVEQKWWCQMQPCLTAFA2Frog Sequence No. 176GQVAGTTRATPSCVDASIVEQKWWCHMQPCLTAFA2Fish Sequence No. 177GQVAGTTRAAPSCVDASIVAQKWWCQMQPCMTAFA3Human, Monkey, Rabbit Sequence No. 178GQVAGTTRAKPSCVDASIVLQRWWCQMEPCLTAFA3Pig, Rat, Mouse Sequence No. 179GQVAGTTRAKPSCVDASIVLQKWWCQMEPCLTAFA3Chicken Sequence No. 180GQVAGTTRAAPSCVDASIVLQKWWCQMEPCLTAFA3Komodo dragon, Fencee lizard sequence number 181GQVAGTTRAAPSCVDASIVLQKWWCQMQPCLTAFA3Wall lizard sequence number 182GQVAGTTHAAPSCVDASIVLQKWWCQMQPCVTAFA3Frog sequence number 183GQVAGTTRAAPSCVDASIVIQKWWCQMEPCLTAFA3Fish sequence number 184GQVAGTTRAAPSCVDASIVAQKWWCQMQPCV

[0440]

[0441] [Example 6] Evaluation of TAFA4 Efficacy in a Mouse Noise-Induced Hearing Loss Model

[0442] The therapeutic efficacy of the TAFA4 protein for hearing loss was evaluated using a mouse noise-induced hearing loss model. Noise-induced hearing loss is a type of sensorineural hearing loss caused by irreversible damage to hair cells due to excessive noise exposure. In the mouse noise-induced hearing loss model, hair cell damage is induced; damage to inner hair cells can be evaluated through Auditory Brainstem Response (ABR) testing, while damage to outer hair cells can be evaluated through Distortion Product Otoacoustic Emission (DPOAE) testing. When inner hair cells are damaged, the process of converting sound stimuli into electrical signals and transmitting them to the auditory nerve is inhibited; consequently, the threshold of the Auditory Brainstem Response, which represents the electrophysiological response of the auditory pathway from the auditory nerve to the brainstem, increases. Outer hair cells act as amplifiers for the cochlea, increasing sensitivity to weak sounds and enabling more precise differentiation of frequency-specific responses in the inner ear. Damage to these outer hair cells can be confirmed by an increase in the threshold in modulated otoacoustic emission testing.

[0443]

[0444] 6.1 Composition of the test group

[0445] Control Group and Test Group Model Administered Substance Route / Frequency Dosage Volume Number of Animals G1 Mock Control AAV Formulation Buffer Unilateral Posterior Recurrent Canal / Single Dose - 1.3 μL 6G2 Noise-Induced Hearing Loss AAV Formulation Buffer Unilateral Posterior Recurrent Canal / Single Dose - 1.3 μL 6G3 Noise-Induced Hearing Loss TAFA4 Overexpressing AAV (AAV8.TAFA4) Unilateral Posterior Recurrent Canal / Single Dose 1 x 10 10 vg1.3 μL8

[0446]

[0447] 6.2 Posterior semicircular canal administration

[0448] AAV formulation buffers (G1 and G2) or AAV (G3: AAV8.TAFA4) were delivered to the cochlea of ​​pups (2–3 days old, Vital River Laboratory Animal Technology Co., Ltd (Pinghu, China)) via posterior semicircular canal administration. On the day of administration, the mice were placed on an ice pack for 2–3 minutes to anesthetize them until movement ceased. Under sterile conditions, the anesthetized pups were laid on their sides, disinfection was performed, and a skin incision approximately 3 mm in length was made behind the ear. The sternocleidomastoid muscle and the adipose tissue above it were gently grasped to expose the posterior semicircular canal. After connecting a microsyringe filled with AAV formulation buffer (G1 or G2) or AAV (G3) to a microsyringe pump controller (WPI Micro 4), the posterior semicircular canal was punctured with the tip of a microfilament assembly (MFA, Microlumen. ID 0.0038" * OD 0.0048") connected to the microsyringe, and a total of 1.3 uL was injected at a rate of 200 nL per minute. After waiting 1 minute to allow the administered substance to spread evenly, the microfilament assembly was removed using a soft cloth under light pressure. The soft tissue was returned to its original position, and the skin incision was sutured using tissue adhesive. To aid in the recovery of the puppies after surgery, they were wrapped in a cloth and placed on a warming pad; once their body temperature stabilized within the normal range, their skin color returned to pink, and their mobility was restored, they were returned to their mother.

[0449]

[0450] 6.3 Model of Noise-Induced Hearing Loss

[0451] Twenty-one days after posterior semicircular canal administration, mice were individually housed in cages installed inside a soundproof chamber. To induce noise, a high-frequency speaker (BM HG10044XT) was mounted directly above the cage and connected to a digital signal processing system (Tucker-Davis Technologies) and an amplifier (Yamaha PX3). Mice were exposed to noise with a frequency of 8–16 kHz and an intensity of 105 dB SPL for two hours, and the acoustic stimulus was calibrated using a sound level meter prior to the experimental session. After noise exposure, the mice were returned to their original cages for recovery, and care was taken to minimize handling stress during the transfer process.

[0452]

[0453] 6.4 Auditory Brainstem Response Test

[0454] Twenty-four days after posterior semicircular canal administration, animals were anesthetized using xylazine (16 mg / kg, 2 mL / kg, intraperitoneal) and zolletil (40 mg / kg, 2 mL / kg, intraperitoneal). The anesthetized animals were placed in a lateral decubitus position inside a soundproof chamber, and body temperature was maintained using a temperature-controlled heating pad. Three subcutaneous needle electrodes were positioned with the recording electrode at the parietal region, the reference electrode at the mastoid process, and the ground electrode on the ipsilateral hind leg; it was confirmed that the impedance of all electrodes was less than 1 kΩ. Acoustic stimulation was provided using a 5 ms tone pip (0.5 ms rise / fall time, cos² envelope) at a frequency of 16 kHz, presented 40 times per second. Stimulation intensity started at 90 dB SPL and decreased in 5 dB increments until 20 dB SPL was reached or the reproducible waveform shape was lost. The threshold was recorded at the point where at least three consistent waveform features (waves I-III) maintaining a latency-intensity relationship were confirmed. All electrodes were removed after the recording was completed. Statistical significance was confirmed via one-way ANOVA.

[0455] It was confirmed that the auditory brainstem response threshold was statistically significantly increased in the noise-induced hearing loss group (G2) compared to the control group (G1) (****p< 0.0001) (Fig. 7). In the TAFA4 overexpressing AAV administration group (G3), a tendency for the auditory brainstem response threshold to decrease was observed compared to the noise-induced hearing loss group (G2). These results suggest that TAFA4 may play a protective role against inner hair cell function damage caused by noise.

[0456]

[0457] 6.5 Modulated Otoacoustic Emission Test

[0458] After the auditory brainstem response test, modulated otoacoustic emissions were subsequently measured in the same ear.

[0459] A specialized probe equipped with a dual acoustic driver and microphone / preamplifier assembly (TDT RZ6 / BioSigRZ system) was precisely positioned at the entrance of the external auditory canal using a micro-manipulator. The stimulus utilized a primary tone with a frequency ratio of f2 / f1 = 1.2, and the intensity was set to L1 = L2 + 10 dB. The test was conducted at a frequency of 16 kHz. The lowest stimulus intensity at which a response higher than 10 dB SPL relative to the noise floor was observed, as confirmed by spectral averaging, was recorded as the modulated otoacoustic emission threshold. After the test, the animals were transferred to a thermal recovery chamber and observed until full walking ability was restored, after which they were returned to their original cages. Statistical significance was confirmed via one-way ANOVA.

[0460] It was confirmed that the modulated otoacoustic emission threshold was statistically significantly increased in the noise-induced hearing loss group (G2) compared to the control group (G1) (***p< 0.001) (Fig. 8). In the TAFA4 overexpressing AAV administration group (G3), it was confirmed that the threshold increased by noise exposure was reduced to a level similar to that of the control group (G1) (ns vs G1, **p< 0.01 vs G2). These results suggest that TAFA4 effectively inhibits outer hair cell function damage caused by noise.

[0461]

[0462] All publications, patents, patent applications, and other documents cited in this application are likewise incorporated herein by reference in their entirety for all purposes, even if each individual publication, patent, patent application, or other document is indicated as being included by reference individually for all purposes.

[0463]

[0464] Although the present disclosure has been described above with reference to the embodiments described above, those skilled in the art will understand that various modifications and changes are possible by adding, changing, deleting, or inserting components without departing from the spirit of the present disclosure as set forth in the claims. It will be understood that such modifications and changes are within the scope of the present disclosure.

Claims

1. A pharmaceutical composition for the prevention or treatment of inner ear disease, comprising: (i) a polypeptide comprising an amino acid sequence of one or more proteins selected from the group consisting of TAFA1 to TAFA4 proteins or fragments thereof, or variants thereof; a nucleic acid molecule encoding said polypeptide; a vector comprising said nucleic acid molecule; a recombinant virus particle comprising said vector and a capsid protein; a cell comprising said vector; a cell transformed with said vector; or a combination thereof; and (ii) a pharmaceutically acceptable carrier.

2. A pharmaceutical composition for the prevention or treatment of a disease caused by damage or abnormality of ribbon synapses in the inner ear, comprising: (i) a polypeptide comprising an amino acid sequence of one or more proteins selected from the group consisting of TAFA1 to TAFA4 proteins or fragments thereof, or variants thereof; a nucleic acid molecule encoding said polypeptide; a vector comprising said nucleic acid molecule; a recombinant virus particle comprising said vector and a capsid protein; a cell comprising said vector; a cell transformed with said vector; or a combination thereof; and (ii) a pharmaceutically acceptable carrier.

3. A pharmaceutical composition according to paragraph 2, wherein the disease caused by damage or abnormality of the ribbon synapse is an inner ear disease.

4. A pharmaceutical composition according to paragraph 2, wherein the damage or abnormality of the ribbon synapses comprises a decrease in the number of ribbon synapses or abnormal function of the ribbon synapses compared to a corresponding subject without damage to the ribbon synapses in the inner ear.

5. In paragraph 2, the pharmaceutical composition is a pharmaceutical composition that induces ribbon synapse formation in the inner ear of a subject administered the same or improves ribbon synapse function.

6. A pharmaceutical composition according to claim 5, wherein the function comprises the ability to release one or more neurotransmitters in response to a signal from an inner hair cell.

7. A pharmaceutical composition according to claim 5, wherein the number of ribbon synapses in the inner ear of the subject increases after administration compared to the subject before administration or the corresponding subject not administered.

8. A pharmaceutical composition according to claim 1 or 3, wherein the inner ear disease is caused by i) a disease or dysfunction of the auditory system in the cochlea or ii) a disease or dysfunction of the balance system in the vestibular system.

9. A pharmaceutical composition according to claim 8, wherein the auditory system disease or dysfunction is hidden hearing loss, noise-induced synaptopathy, age-related synaptopathy, tinnitus, sensorineural hearing loss (SNHL), or a combination thereof.

10. A pharmaceutical composition according to claim 9, wherein the sensorineural hearing loss may or may not be accompanied by tinnitus.

11. A pharmaceutical composition according to claim 9, wherein the sensorineural hearing loss comprises hereditary or non-hereditary sensorineural hearing loss.

12. A pharmaceutical composition according to claim 11, wherein the non-hereditary sensorineural hearing loss comprises ototoxic hearing loss, noise-induced hearing loss (NIHL), hearing loss due to infection, hearing loss due to immune abnormality or inflammation, hearing loss due to trauma, presbycusis, sudden sensorineural hearing loss (SSNHL), endolymphatic hydrops, or a combination thereof.

13. A pharmaceutical composition according to claim 11, wherein the hereditary sensorineural hearing loss comprises a hereditary sensorineural hearing loss selected from the group consisting of nonsyndromic hearing loss (Nonsyndromic HHL) and syndromic hearing loss (Syndromic HHL).

14. A pharmaceutical composition according to claim 13, wherein the syndromic hearing loss is an auditory neuropathy spectrum disorder, Usher syndrome, Pendred syndrome, Waardenburg syndrome, Alport syndrome, Branchio-oto-renal (BOR) syndrome, CHARGE syndrome, Jervell and Lange-Nielsen syndrome, Stickler syndrome, Treacher Collins syndrome, a mitochondrial genetic disorder, or a combination thereof.

15. A pharmaceutical composition according to claim 1, wherein the inner ear disease is caused by damage to the spiral ganglion, degeneration of the nerve processes, or branching disorder.

16. The pharmaceutical composition of claim 1, wherein the pharmaceutical composition increases the growth or branching points of the neurites of the spiral ganglion neurons of the subject administered with the same.

17. A pharmaceutical composition according to claim 15, wherein the inner ear disease is Auditory Neuropathy Spectrum Disorder (ANSD), Noise-induced hearing loss, Age-related hearing loss (ARHL), Diabetic Auditory Neuropathy, Spiral Ganglionitis, Vestibular Schwannoma, or a combination thereof.

18. A pharmaceutical composition according to claim 1 or 2, wherein the polypeptide comprises an amino acid sequence (from the N-terminus to the C-terminus) of the following general formula 7: <General Formula 7> X1-X2-X3-GTCEV-X4-A-X5-H-X6- CCN-X7-N-X8-IEE- -X20-X21-X22-WWC-X23-M-X24-PC-X25-X26-GE-X27-CK-X28-LPD-X29-X30-GW-X31-C-X32-X33-G-X34-K-X35-KTT-X36-X37-X38-X39 In the above general formula 7, X1 does not exist, or is V, I, or L, X2 is K, E, R, or Q, and X3 is G, T, Q, P or A, and X4 is V or I, and X5 is A, L, V or I, and X6 is R or L, and X7 is K, R, or Q, and X8 is R or K, and X9 is R or L, and X10 is V or G, and X11 is K or N, and X12 is F or L, and X13 is P or S, and X14 is Q or K, and X15 is R, H, or Q, and X16 is A, N, S or T, and X17 is A, Q, R, K or T, and X18 is D or E, and X19 is S or A, and X20 is I, E, L, A, or V, and X21 is Q, G, or E, and X22 is K or R, and X23 is H, Q, or E, and X24 is E, Q, N, D, S or H, and X25 is L, V, or M, and X26 is E, D, P, L or A, and X27 is E or D, and X28 is V, T, A or I, and X29 is L, N, R, Y, S or Q, and X30 is S, K, or T, and X31 is S or M, and X32 is S, A, or Y, and X33 is S, T, or R, and X34 is N or H, and X35 is V or I, X36 is R or K, and X37 does not exist or is V, A, G, M, or N, X38 does not exist or is T, I, N, F, or S, and X39 is either non-existent or R, H, V, K, I, or Q.

19. A pharmaceutical composition according to claim 18, wherein the polypeptide comprises one or more amino acid sequences selected from the group consisting of amino acid sequences presented in SEQ ID NOs 87 to 141.

20. In claim 1 or 2, the polypeptide is (i) composed of a sequence of 8 to 61 amino acids, and (ii) A pharmaceutical composition characterized by comprising a sequence having at least 50% sequence identity with the amino acid sequence of SEQ ID NO. 1 or the amino acid sequence of SEQ ID NO.

15.

21. A pharmaceutical composition according to claim 20, wherein the polypeptide comprises an amino acid sequence of the following general formula 1 (from the N-terminus to the C-terminus): <General Formula 1> X1-GE-X2-CK-X3-L In the above general formula 1 X1 is E, D, P, L or A, and X2 is D or E, and X3 is T, V, I, or A.

22. A pharmaceutical composition according to claim 21, characterized in that the polypeptide comprises the amino acid sequence of SEQ ID NO.

142.

23. A pharmaceutical composition according to claim 21, wherein the polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs 1 to 14.

24. A pharmaceutical composition according to claim 21, characterized in that the polypeptide consists of a sequence of 8 to 43 amino acids.

25. A pharmaceutical composition according to claim 20, wherein the polypeptide comprises an amino acid sequence of the following general formula 2 (from the N-terminus to the C-terminus): <General Formula 2> IV-X4-X5-X6-WWC-X7-M-X8-PC-X9-X1-GE-X2-CK-X3-L In the above general formula 2 X1 is E, D, P, L or A, and X2 is D or E, and X3 is T, V, I or A, and X4 is I, E, A, L or V, and X5 is Q, E, or G, and X6 is K or R, and X7 is E, H, or Q, and X8 is E, Q, N, D, S or H, and X9 is L, M, or V.

26. A pharmaceutical composition according to claim 25, characterized in that the polypeptide comprises the amino acid sequence of SEQ ID NO.

143.

27. A pharmaceutical composition according to claim 25, wherein the polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs 28 to 51.

28. A pharmaceutical composition according to claim 20, wherein the polypeptide comprises the amino acid sequence of the following general formula 3 (from the N-terminus to the C-terminus): <General Formula 3> X1-GE-X2-CK-X3-LPD-X4-X5-GWSCS-X6-GNK-X7-KTTKVTR In the above general formula 3 X1 is E, D, P, L or A, and X2 is D or E, and X3 is T, V, I or A, and X4 is Y, S, or L, and X5 is S or T, and X6 is S or T, and X7 is V or I.

29. A pharmaceutical composition according to claim 28, characterized in that the polypeptide comprises the amino acid sequence of SEQ ID NO.

144.

30. A pharmaceutical composition according to claim 28, characterized in that the polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs 52 to 58.

31. A pharmaceutical composition according to claim 20, characterized in that the polypeptide comprises the amino acid sequence of the following general formula 4 (from the N-terminus to the C-terminus): <General Formula 4> G-X1-VAGTT-X2-X3-X4-PSCV-X5-X6-X7 In the above general formula 4 X1 is Q or K, and X2 is R, H, or Q, and X3 is A, N, S or T, and X4 is R, A, Q, K, or T, and X5 is D or E, and X6 is A or does not exist, and X7 is S, A, or does not exist.

32. A pharmaceutical composition according to claim 31, characterized in that the polypeptide comprises the amino acid sequence of SEQ ID NO.

145.

33. A pharmaceutical composition according to claim 31, wherein the polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs 15 to 27.

34. A pharmaceutical composition according to claim 31, characterized in that the polypeptide consists of a sequence of 15 to 46 amino acids.

35. A pharmaceutical composition according to claim 20, characterized in that the polypeptide comprises the amino acid sequence of the following general formula 5 (from the N-terminus to the C-terminus): <General Formula 5> X8-IEE-X9-SQT-X10-X11-CSC-X12-X13-G-X1-VAGTT-X2-X3-X4-PSCV-X5-X6-X7 In the above general formula 5 X1 is Q or K, and X2 is R, H, or Q, and X3 is A, N, S or T, and X4 is R, A, Q, K, or T, and X5 is D or E, and X6 is A or does not exist, X7 is S, A, or does not exist, X8 is R or K, and X9 is R or L, and X10 is V or G, and X11 is K or N, and X12 is F or L, and X13 is P or S.

36. A pharmaceutical composition according to claim 35, characterized in that the polypeptide comprises the amino acid sequence of SEQ ID NO.

146.

37. A pharmaceutical composition according to claim 35, wherein the polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs 59 to 74.

38. A pharmaceutical composition according to claim 20, wherein the polypeptide comprises the amino acid sequence of the following general formula 6 (from the N-terminus to the C-terminus): <General Formula 6> G-X1-VAGTT-X2-X3-X4-PSCV-X5-X6-X7-IV-X8-X9-KWWC-X10-M-X11-PC-X12 In the above general formula 6 X1 is Q or K, and X2 is R, H, or Q, and X3 is A, N, S or T, and X4 is R, A, Q, K or T, and X5 is D or E, and X6 is A or does not exist, X7 is S, A, or does not exist, X8 is I, A, V or L, and X9 is Q or E, and X10 is H or Q, and X11 is N, D, S or H, and X12 is L or M.

39. A pharmaceutical composition according to claim 38, characterized in that the polypeptide comprises the amino acid sequence of SEQ ID NO.

147.

40. A pharmaceutical composition according to claim 38, wherein the polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs 75 to 85.

41. A method for preparing a pharmaceutical composition for the prevention or treatment of inner ear disease, comprising the step of preparing a composition comprising: a polypeptide comprising an amino acid sequence of one or more proteins selected from the group consisting of TAFA1 to TAFA4 proteins, a fragment thereof, or a variant thereof; a nucleic acid molecule encoding said polypeptide; a vector comprising said nucleic acid molecule; a recombinant virus particle comprising said vector and a capsid protein; a cell comprising said vector; a cell transformed with said vector; or a combination thereof.

42. Use for the prevention or treatment of inner ear disease for the manufacture of a drug comprising: a polypeptide comprising an amino acid sequence of one or more proteins selected from the group consisting of TAFA1 to TAFA4 proteins or fragments thereof, or variants thereof; a nucleic acid molecule encoding said polypeptide; a vector comprising said nucleic acid molecule; a recombinant virus particle comprising said vector and a capsid protein; a cell comprising said vector; a cell transformed with said vector; or a combination thereof.

43. Use for the prevention or treatment of a disease caused by inner ear ribbon synapse damage for the manufacture of a drug comprising: a polypeptide comprising an amino acid sequence of one or more proteins selected from the group consisting of TAFA1 to TAFA4 proteins or fragments thereof, or variants thereof; a nucleic acid molecule encoding said polypeptide; a vector comprising said nucleic acid molecule; a recombinant virus particle comprising said vector and a capsid protein; a cell comprising said vector; a cell transformed with said vector; or a combination thereof.

44. A method for treating an inner ear disease in a subject requiring treatment, comprising administering to the subject a composition comprising: a polypeptide comprising an amino acid sequence of one or more proteins selected from the group consisting of TAFA1 to TAFA4 proteins, a fragment thereof, or a variant thereof; a nucleic acid molecule encoding said polypeptide; a vector comprising said nucleic acid molecule; a recombinant virus particle comprising said vector and a capsid protein; a cell comprising said vector; a cell transformed with said vector; or a combination thereof.

45. A method for treating a disease caused by damage to the inner ear ribbon synapse in a subject requiring treatment, comprising administering to the subject a composition comprising: a polypeptide comprising an amino acid sequence of one or more proteins selected from the group consisting of TAFA1 to TAFA4 proteins, a fragment thereof, or a variant thereof; a nucleic acid molecule encoding said polypeptide; a vector comprising said nucleic acid molecule; a recombinant virus particle comprising said vector and a capsid protein; a cell comprising said vector; a cell transformed with said vector; or a combination thereof.

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