Zinc finger protein for treating charcot-marie-tooth disease and method for expressing same

Abstract

The present invention relates to a system for regulating overexpression of PMP22 gene by a P1 promoter in patients with Charcot-Marie Tooth disease (CMT) to a normal level by using zinc finger protein (ZFP). Specifically, the present invention provides an isolated ZFP consisting of three or more zinc fingers and specifically reducing the expression of a PMP22 protein by a P1 promoter by binding to a promoter of the PMP22 gene or a part thereof in nervous system cells, or a polynucleotide encoding same. The present invention also provides novel transcriptional regulatory sequences for Schwann cell-specific expression of nucleic acid molecules encoding genes to be expressed in Schwann cells or proteins to be expressed in Schwann cells. The ZFP for regulating PMP22 gene expression according to the present invention or a polynucleotide encoding same can be used as a therapeutic agent for preventing or treating CMT disease, and can be more effectively expressed by the Schwann cell-specific transcriptional regulatory sequence.

Description

Zinc finger proteins for treating Charcot-Marie-Tooth disease and methods for expressing them

[0001] The present invention relates to a system for regulating PMP22 gene overexpression to a normal level in patients with Schwann cell-related diseases, particularly Charcot-Marie-Tooth (CMT) disease, using a zinc finger protein (ZFP). Specifically, an isolated ZFP or a polynucleotide encoding the same is provided, which comprises three or more zinc finger motifs and binds to the promoter of the PMP22 gene or a portion thereof in nervous system cells to reduce the expression of the PMP22 protein. The PMP22 gene expression-regulating ZFP according to the present invention or the polynucleotide encoding the same can be used as a therapeutic agent for preventing or treating CMT disease. Accordingly, the present invention provides a use of the above substance for preventing or treating CMT disease.

[0002] The present invention also relates to a novel transcriptional regulatory sequence for increased expression of a nucleic acid molecule encoding a gene to be expressed in Schwann cells or a protein to be expressed in Schwann cells, a polynucleotide comprising the same, or a vector (e.g., an expression vector) comprising the same. The transcriptional regulatory sequence according to the present invention is useful for the expression of a protein capable of suppressing the expression of a gene overexpressed in a Schwann cell-related disease, and is particularly suitable for expressing a ZFP capable of reducing the expression of the PMP22 gene overexpressed in CMT disease in Schwann cells. The transcriptional regulatory sequence or expression vector according to the present invention can be used for the prevention, improvement, or treatment of diseases related to Schwann cells, particularly CMT disease. Accordingly, the present invention provides a method for expressing a ZFP useful for the treatment of Charcot-Marie-Tooth disease using the novel transcriptional regulatory sequence described above.

[0003] CMT disease, also known as hereditary motor and sensory neuropathy, is a disease characterized by progressive sensory impairment and muscle weakness. CMT disease is one of the most common inherited peripheral neuropathy, affecting approximately 1 in 2,500 people worldwide, and more than 80 causative genes are known. CMT disease is characterized by progressive muscle weakness and atrophy, primarily in the lower extremities, progressing proximally. Symptoms may also include areflexia, distal sensory loss, foot deformities (pes cavus), and hearing impairment. The disease typically develops in the teen years, but can also occur in individuals in their 30s or older.

[0004] CMT disease type 1A (CMT1A) is one of the major types of CMT disease, and the pathogenic factor of CMT1A is known to be an increase in the number of PMP22 gene copies due to duplication of chromosome 17p11.2, and PMP22 overexpression causes abnormal myelination of Schwann cells, causing peripheral nerve disease. Transcription of the PMP22 gene is mainly mediated by promoter 1 (P1) and promoter 2 (P2), and shows a different expression pattern between exon 1a and exon 1b, and it is known that PMP22 mRNA expression induced by the P1 promoter is high in the sciatic nerve and Schwann cells.

[0005] [Prior Art Literature]

[0006] (Patent Document)

[0007] Patent Publication No. 10-2023-0110194

[0008] Despite extensive research into the development of therapeutics for Schwann cell-related diseases, particularly CMT, in the fields of small molecule compounds, cell therapy, and gene therapy, there remains a fundamental lack of treatment options due to the lack of approved therapeutics. Furthermore, for the effective application of gene therapy to treat diseases caused by Schwann cell dysfunction, such as CMT, there is a need for specific and increased expression of gene therapy agents in Schwann cells. Therefore, the present invention provides a substance useful for preventing or treating CMT.

[0009] Another object of the present invention is to provide a novel transcriptional regulatory sequence capable of increasing the expression of a substance that regulates transcription of the PMP22 gene causing CMT disease and a substance useful for treating diseases related to Schwann cells, the expression of which is required in Schwann cells, and thereby providing a method for effectively expressing a substance capable of preventing or treating CMT disease. In addition, the present invention is to provide an expression vector or pharmaceutical composition that can increase the expression of ZFP capable of reducing the expression of the PMP22 gene by using the transcriptional regulatory sequence, and thereby can be useful for preventing, improving or treating CMT disease, or a CMT treatment method using the same.

[0010] The present invention provides an isolated ZFP or a polynucleotide encoding the same, which comprises at least three zinc finger motifs and binds to the promoter of the PMP22 gene or a portion thereof in a nervous system cell to reduce the expression of the PMP22 protein, and a method for preventing or treating a disease associated with Schwann cells, particularly CMT disease, using the same. For example, the ZFP or the polynucleotide encoding the same according to the present invention can be administered to a CMT patient by being contained in a plasmid, a viral vector, or a non-viral vehicle. Prior to the present invention, there was no known method for preventing or treating CMT disease by inhibiting transcription of the PMP22 gene through ZFP in a CMT patient.

[0011] The present invention also provides a transcriptional regulatory sequence of a novel nucleotide sequence. The transcriptional regulatory sequence is operably linked to a nucleic acid molecule encoding a gene to be expressed in Schwann cells or a protein to be expressed in Schwann cells, and can be used in the treatment of diseases associated with Schwann cells. The target gene or nucleic acid molecule may be a nucleic acid encoding ZFP, which binds to the promoter or a portion thereof of the PMP22 gene in nervous system cells and reduces the expression of the PMP22 protein. The present invention provides a polynucleotide comprising the transcriptional regulatory sequence according to the present invention and a ZFP-encoding nucleic acid, or a carrier (e.g., an expression vector) comprising the same, and the carrier can be used to treat CMT disease. In this respect, the present invention also provides a pharmaceutical composition for treating CMT disease and a method for treating CMT disease using the same.

[0012] The transcriptional regulatory sequence according to the present invention is suitable for expression in Schwann cells and for gene regulation required for the treatment of diseases associated with Schwann cells. In particular, it is suitable for expressing ZFP, which binds to the promoter of the PMP22 gene and reduces the expression of the PMP22 protein, in Schwann cells. Therefore, the present invention is useful for the treatment of CMT disease, which is characterized by overexpression of the PMP22 gene.

[0013] Figure 1 shows the DNA target sites of ZFPs screened for their PMP22 gene transcription repression activity using an in vitro reporter system. The region from -10 bp to +30 bp from the TATA box of the P1 promoter of the PMP22 gene to the transcription start site was selected as the ZFP target site. The sequences shown for each ZFP represent the DNA sequences recognized by the ZFP (recognition sequences of the upper strand or bottom strand of the target PMP22 gene sequence). The outlined rectangles represent 4-zinc finger motif, 5-zinc finger motif, or 6-zinc finger motif proteins, respectively, and the black-shaded nucleotides represent 1 bp skipped sites.

[0014] Figure 2 shows the results of measuring the PMP22 gene transcription repression activity of the ZFPs described in Figure 1 using an in vitro reporter system. HEK239T represents the results obtained from HEK239T cells, and sNF02.2 represents the results obtained using sNF02.2 cells, a Schwann-like cell line.

[0015] Figure 3 shows the PMP22 gene transcription inhibitory activity of ZFP according to the present invention confirmed through analysis of PMP22 transcript expression levels in Schwann-like cells. The experimental results show that ZFP according to the present invention specifically inhibits PMP22 gene expression induced by the P1 promoter without substantially affecting PMP22 gene expression induced by the P2 promoter.

[0016] Figure 4 shows the results of confirming that the ZFP according to the present invention binds effectively to the PMP22 P1 promoter when expressed using the CUT & RUN analysis technique, through a flag tag linked to the ZFP protein. IgG was used as a negative control to confirm the specific binding of the anti-flag antibody used to the ZFP protein.

[0017] Figure 5 shows the results of measuring the transcriptional regulatory activity of the Schwann cell transcriptional regulatory sequence according to the present invention through flow cytometry.

[0018] Figure 6 shows the results of confirming the expression of a vector containing a Schwann cell transcription control sequence according to the present invention together with a ZFP encoding sequence.

[0019] Figure 7 shows the results of measuring the transcriptional regulation efficiency of the PMP22 gene using a ZFP expression vector including a Schwann cell transcriptional regulatory sequence according to the present invention. This experimental result shows that when the ZFP and Schwann cell transcriptional regulatory sequence according to the present invention are used, the expression of the PMP22 gene induced by the P2 promoter of the PMP22 gene is not substantially affected, but the expression of the PMP22 gene induced by the P1 promoter is specifically suppressed.

[0020] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Generally, the terms used herein are those well known and commonly used in the art.

[0021] The term “sequence” in this specification may be interpreted, depending on the context, as a nucleic acid (or polynucleotide) molecule or a protein (or polypeptide) molecule having a given sequence.

[0022] As used herein, the terms "target" or "target site" refer to a pre-identified nucleic acid sequence of any composition and / or length. Such target sites include, but are not limited to, genes, promoters, or any nucleic acid sequence.

[0023] The present invention provides an isolated ZFP comprising at least three zinc finger motifs and binding to the promoter or a portion thereof of the PMP22 gene in a nervous system cell to reduce the expression of the PMP22 protein. The promoter is preferably the P1 promoter.

[0024] The above PMP22 (Peripheral myelin protein 22) is also called Growth arrest-specific protein 3 (GAS-3), and the PMP22 gene encodes a 22 kDa transmembrane glycoprotein consisting of 160 amino acids that is mainly expressed in Schwann cells of the peripheral nervous system. In humans, the PMP22 gene may consist of the nucleotide sequence of SEQ ID NO: 377, is located on chromosome 17p11.2, and is approximately 40 kb in length. The gene contains six exons that are conserved in both humans and rodents, two of which are 5' untranslated exons (1a and 1b), and produce two different RNA transcripts with the same coding sequence.

[0025] The PMP22 gene can cause dysfunction of protein synthesis and myelin sheath due to inappropriate intracellular levels. Changes in the expression of the PMP22 gene have been associated with various neuropathies, such as CMT, Dejerine-Sottas disease, and Hereditary Neuropathy with Liability to Pressure Palsy (HNPP). For example, overexpression of PMP22 (gene duplication) is known to cause CMT, and reduced expression of PMP22 (due to gene deletion, etc.) is known to cause HNPP.

[0026] The 5'-UTR of the PMP22 gene contains two known promoters, P1 and P2. Their respective transcripts differ only in their 5' non-coding region [Bosse et al., J. Neuroscience Res., 37(4): 529-537 (1994) and Suter et al., J. Biol. Chem., 269(41): 25795-25808 (1994)], but give rise to six splice variants that exhibit tissue-specific expression patterns [Visigalli et al., Hum. Mut., 37(1): 98-109 (2015)]. PMP22 regulation is achieved through the intronic region and enhancer elements within it [Jones et al., Hum. Mol. Genet., 21(7): 1581-1591 (2012); Srinivasan et al., Nuc. Acids Res., 40(14): 6449-6460 (2012); and Pantera et al., Hum. Mol. Genet., 27(16): 2830-2839 (2018)]. The P1 promoter transcript is Schwann cell-specific, whereas the P2 promoter transcript is expressed in non-PNS tissues. Therefore, duplication of the PMP22 gene or its major transcriptional binding site alters the ratio of splicing isoforms and alters methylation, microRNA binding, and posttranslational modification sites [Verrier et al., Glia, 57(12): 1265-1279 (2009) and Lee et al., Exp. Neurobiology, 28(2): 279-288 (2019)]. In normal myelinated and non-myelinated Schwann cells, approximately 20% of newly synthesized PMP22 is glycosylated, and the remaining ~80% is targeted for proteasomal endoplasmic reticulum (ER)-associated degradation (ERAD).

[0027] ZFP

[0028] A "zinc finger protein," referred to herein as a "ZFP," refers to a protein that binds DNA in a sequence-specific manner via one or more zinc finger motifs, or a portion of a larger protein that binds DNA. In the present invention, the ZFP preferably comprises, but is not necessarily limited to, three to six zinc finger motifs.

[0029] A zinc finger motif is a small polypeptide domain of approximately 20-40 amino acid residues, in which four amino acid residues, cysteine ​​and / or histidine, are appropriately arranged to form a coordination bond with a zinc ion. Depending on the type of residue that forms a coordination bond with the zinc ion, the zinc finger motif can be classified into, for example, C2H2 (Cys2-His2), C3H (Cys2-CysHis), C4 (Cys2-Cys2), etc. The zinc finger motif used in the present invention is preferably a C2H2 zinc finger. The term "zinc finger motif" as used herein may also be referred to as "zinc finger" or "zinc finger domain".

[0030] The zinc finger motif used in the ZFP according to the present invention may be a natural zinc finger naturally occurring in any eukaryote, including humans, or a variant thereof, or may be an artificially produced one. Meanwhile, the ZFP according to the present invention is an artificially produced one in that it is a combination and / or rearrangement of such zinc finger motifs, and the entire sequence is an amino acid sequence that does not exist in nature.

[0031] The ZFP according to the present invention can preferably recognize a part of the sequence (5'-CCAGCCCCTGAATAAACTGGAAAGACGCCTGGTCTGGCTTCAGTTACAGGGAGCACCACCAGGGAACATC-3' (SEQ ID NO: 1)) from -10 bp to +30 bp based on the transcription initiation site from the TATA box of the P1 promoter of the PMP22 gene. In addition, it can recognize a part of the complementary sequence of the above sequence (5'-GATGTTCCCTGGTGGTGCTCCCTGTAACTGAAGCCAGACCAGGCGTCTTTCCAGTTTATTCAGGGGCTGG-3' (SEQ ID NO: 2)).

[0032] The ZFP according to the present invention suppresses the expression of the PMP22 protein induced by the P1 promoter of the PMP22 gene. Whether the expression of the PMP22 protein is suppressed by the ZFP can be determined by comparing the expression level of the PMP22 gene transcript with that of the control when the ZFP is expressed, but is not necessarily limited to this method. Preferably, the ZFP according to the present invention specifically suppresses the expression of the PMP22 protein induced by the P1 promoter of the PMP22 gene compared to the P2 promoter of the PMP22 gene. Specifically suppressing the expression of the PMP22 protein induced by the P1 promoter compared to the P2 promoter of the PMP22 gene means suppressing the expression of the PMP22 protein induced by the P1 promoter without substantially affecting the expression of the PMP22 gene induced by the P2 promoter. For example, it can be measured by quantifying the expression of P1 promoter transcripts and P2 promoter transcripts of the PMP22 gene by designing primers to distinguish between transcripts induced by the P1 promoter and transcripts induced by the P2 promoter, but is not necessarily limited to this method. The expression that it does not substantially affect PMP22 gene expression can mean that the PMP22 gene expression rate when ZFP is expressed does not show a statistically significant difference when compared to the control group, or is 10% or less or 5% or less compared to the average value of the PMP22 gene expression rate obtained from the control group.

[0033] The ZFP according to the present invention may bind to a nucleic acid sequence portion selected from the following group or a complementary sequence thereof.

[0034] Number Target Sequence 15'-CCAGCCCCTGAA-3' (SEQ ID NO: 3) 25'-ATAAACTGGAAA-3' (SEQ ID NO: 4) 35'-TGGAAAGACGCC-3' (SEQ ID NO: 5) 45'-AAAGACGCCTGG-3' (SEQ ID NO: 6) 55'-CAGGGAGCACCA-3' (SEQ ID NO: 7) 65'-AGGGAGCACCAC-3' (SEQ ID NO: 8) 75'-GCACCACCAGGG-3' (SEQ ID NO: 9) 85'-CACCACCAGGGA-3' (SEQ ID NO: 10) 95'-CCACCAGGGAAC-3' (SEQ ID NO: 11) 105'-CACCAGGGAACA-3' (SEQ ID NO: 12) 115'-CCTGGTGGTGCT-3' (SEQ ID NO: 13)125'-CCTGTAACTGAA-3' (SEQ ID NO: 14)135'-GTAACTGAAGCC-3' (SEQ ID NO: 15)145'-ACTGAAGCCAGA-3' (SEQ ID NO: 16)155'-GAAGCCAGACCA-3' (SEQ ID NO: 17)165'-AAGCCAGACCAG-3' (SEQ ID NO: 18)175'-GCCAGACCAGGC-3' (SEQ ID NO: 19)185'-CCAGACCAGGCG-3' (SEQ ID NO: 20)195'-AGACCAGGCGTC-3' (SEQ ID NO: 21)205'-TGAATAAACTGGAAA-3' (SEQ ID NO: 22)215'-ATAAACTGGAAAGAC-3' (SEQ ID NO: 23)225'-AACTGGAAAGACGCC-3' (SEQ ID NO: 24) 235'-TGGAAAGACGCCTGG-3' (SEQ ID NO: 25) 245'-CAGGGAGCACCACCA-3' (SEQ ID NO: 26) 255'-AGGGAGCACCACCAG-3' (SEQ ID NO: 27) 265'-GGAGCACCACCAGGG-3' (SEQ ID NO: 28) 275'-GAGCACCACCAGGGA-3' (SEQ ID NO: 29) 285'-GCACCACCAGGGAAC-3' (SEQ ID NO: 30) 295'-CACCACCAGGGAACA-3' (SEQ ID NO: 31) 305'-CCTGTAACTGAAGCC-3' (SEQ ID NO: 32) 315'-GTAACTGAAGCCAGA-3' (SEQ ID NO: 33) 325'-ACTGAAGCCAGACCA-3' (SEQ ID NO:34)335'-GAAGCCAGACCAGGC-3' (SEQ ID NO: 35)345'-AAGCCAGACCAGGCG-3' (SEQ ID NO: 36)355'-GCCAGACCAGGCGTC-3' (SEQ ID NO: 37)365'-TGAATAAACTGGAAAGAC-3' (SEQ ID NO: 38)375'-ATAAACTGGAAAGACGCC-3' (SEQ ID NO: 39)385'-AACTGGAAAGACGCCTGG-3' (SEQ ID NO: 40)395'-CAGGGAGCACCACCAGGG-3' (SEQ ID NO: 41)405'-AGGGAGCACCACCAGGGA-3' (SEQ ID NO: 42)415'-GGAGCACCACCAGGGAAC-3' (SEQ ID NO: 43)425'-GAGCACCACCAGGGAACA-3' (SEQ ID NO: 44) 435'-CCTGTAACTGAAGCCAGA-3' (SEQ ID NO: 45) 445'-GTAACTGAAGCCAGACCA-3' (SEQ ID NO: 46) 455'-ACTGAAGCCAGACCAGGC-3' (SEQ ID NO: 47) 465'-GAAGCCAGACCAGGCGTC-3' (SEQ ID NO: 48)475'-CAGGGAGCA-3'485'-CACCAGGGA-3'495'-GGAGCACCA-3'505'-CAGGGAACA-3'515'-GGTGGTGCT-3'525'-CCTGTAACT- 3'535'-CCTGTAACT-3'545'-AAGCCAGAC-3'555'-GTAACTGAA-3'565'-CCAGACCAG-3'575'-ACTGAAGCC-3'585'-GACCAGGCG-3'

[0035] The ZFP according to the present invention may be selected from the group consisting of amino acid sequences comprising a set of three or more zinc finger motif proteins, for example: ZFP1:

[0036] FECKDCGKAFIQKSNLIRHQRTH (SEQ ID NO: 49),

[0037] YKCPECGKSFSTKNSLTEHQRTH (SEQ ID NO: 50),

[0038] YTCSDCGKAFRDKSCLNR HRRTH (SEQ ID NO: 51), and

[0039] YKCPECGKSFSTSHSLTEHQRTH (SEQ ID NO: 52);

[0040] ZFP2:

[0041] YECVQCGKGFTQSSNLITHQRVH (SEQ ID NO: 53),

[0042] FQCKTCQRKFSRSDHLKTHTRTH (SEQ ID NO: 54),

[0043] FQCRICMRNFSDSGNLRVHIRTH (SEQ ID NO: 55), and

[0044] YTCSYCGKSFTQSNTLKQHTRIH (SEQ ID NO: 56);

[0045] ZFP3:

[0046] YTCSDCGKAFRDKSCLNRHRRTH (SEQ ID NO: 57),

[0047] YRCKYCDRSFSDSSNLQRHVRNIH (SEQ ID NO: 58),

[0048] YECVQCGKGFTQSSNLITHQRVH (SEQ ID NO: 59), and

[0049] FQCKTCQRKFSRSDHLKTHTRTH (SEQ ID NO: 60);

[0050] ZFP4:

[0051] FQCKTCQRKFSRSDHLKTHTRTH (SEQ ID NO: 61),

[0052] YTCSDCGKAFRDKSCLNRHRRTH (SEQ ID NO: 62),

[0053] YRCKYCDRSFSDSSNLQRHVRNIH (SEQ ID NO: 63), and

[0054] YECVQCGKGFTQSSNLITHQRVH (SEQ ID NO: 64);

[0055] ZFP5:

[0056] YKCPECGKSFSTSHSLTEHQRTH (SEQ ID NO: 65),

[0057] YKCPDCGKSFSQSSSLIRHQRTH (SEQ ID NO: 66),

[0058] YKCGQCGKFYSQVSHLTRHQKIH (SEQ ID NO: 67), and

[0059] YKCPECGKSFSRADNLTEHQRTH (SEQ ID NO: 68);

[0060] ZFP6:

[0061] YKCPECGKSFSSKKALTEHQRTH (SEQ ID NO: 69),

[0062] YKCPECGKSFSSKKALTEHQRTH (SEQ ID NO: 70),

[0063] YGCHLCGKAFSKSSNLRRHEMIH (SEQ ID NO: 71), and

[0064] FQCKTCQRKFSRSDHLKTHTRTH (SEQ ID NO: 72);

[0065] ZFP7:

[0066] YKCMECGKAFNRRSHLTRHQRIH (SEQ ID NO: 73),

[0067] YKCPECGKSFSTSHSLTEHQRTH (SEQ ID NO: 74),

[0068] YKCPECGKSFSTSHSLTEHQRTH (SEQ ID NO: 75), and

[0069] YKCPDCGKSFSQSSSLIRHQRTH (SEQ ID NO: 76);

[0070] ZFP8:

[0071] YKCGQCGKFYSQVSHLTRHQKIH (SEQ ID NO: 77),

[0072] YKCPECGKSFSRADNLTEHQRTH (SEQ ID NO: 78),

[0073] YKCPECGKSFSSKKALTEHQRTH (SEQ ID NO: 79), and

[0074] YKCPECGKSFSSKKALTEHQRTH (SEQ ID NO: 80);

[0075] ZFP9:

[0076] FQCRICMRNFSDSGNLRVHIRTH (SEQ ID NO: 81),

[0077] YKCMECGKAFNRRSHLTRHQRIH (SEQ ID NO: 82),

[0078] YKCPECGKSFSTSHSLTEHQRTH (SEQ ID NO: 83), and

[0079] YKCPECGKSFSTSHSLTEHQRTH (SEQ ID NO: 84);

[0080] ZFP10:

[0081] YKCPECGKSFSSPADLTRHQRTH (SEQ ID NO: 85),

[0082] YKCGQCGKFYSQVSHLTRHQKIH (SEQ ID NO: 86),

[0083] YKCPECGKSFSRADNLTEHQRTH (SEQ ID NO: 87), and

[0084] YKCPECGKSFSSKKALTEHQRTH (SEQ ID NO: 88);

[0085] ZFP11:

[0086] YECNYCGKTFSVSSTLIRHQRIH (SEQ ID NO: 89),

[0087] YRCEECGKAFRWPSNLTRHKRIH (SEQ ID NO: 90),

[0088] YRCEECGKAFRWPSNLTRHKRIH (SEQ ID NO: 91), and

[0089] YKCPECGKSFSTKNSLTEHQRTH (SEQ ID NO: 92);

[0090] ZFP12:

[0091] FECKDCGKAFIQKSNLIRHQRTH (SEQ ID NO: 93),

[0092] YKCPECGKSFSTHLDLIRHQRTH (SEQ ID NO: 94),

[0093] YKCPDCGKSFSQSSSLIRHQRTH (SEQ ID NO: 95), and

[0094] YKCPECGKSFSTKNSLTEHQRTH (SEQ ID NO: 96);

[0095] ZFP13:

[0096] YTCSDCGKAFRDKSCLNRHRRTH (SEQ ID NO: 97),

[0097] FECKDCGKAFIQKSNLIRHQRTH (SEQ ID NO: 98),

[0098] YKCPECGKSFSTHLDLIRHQRTH (SEQ ID NO: 99), and

[0099] YKCPDCGKSFSQSSSLIRHQRTH(SEQ ID NO: 100);

[0100] ZFP14:

[0101] YECHDCGKSFRQSTHLTQHRRIH (SEQ ID NO: 101),

[0102] YTCSDCGKAFRDKSCLNRHRRTH (SEQ ID NO: 102),

[0103] FECKDCGKAFIQKSNLIRHQRTH (SEQ ID NO: 103), and

[0104] YKCPECGKSFSTHLDLIRHQRTH (SEQ ID NO: 104);

[0105] ZFP15:

[0106] YKCPECGKSFSTSHSLTEHQRTH (SEQ ID NO: 105),

[0107] YECHDCGKSFRQSTHLTQHRRIH (SEQ ID NO: 106),

[0108] YTCSDCGKAFRDKSCLNRHRRTH (SEQ ID NO: 107), and

[0109] FECKDCGKAFIQKSNLIRHQRTH(SEQ ID NO: 108);

[0110] ZFP16:

[0111] YKCPECGKSFSRADNLTEHQRTH (SEQ ID NO: 109),

[0112] YRCKYCDRSFSDSSNLQRHVRNIH (SEQ ID NO: 110),

[0113] YKCPECGKSFSTSHSLTEHQRTH (SEQ ID NO: 111), and

[0114] FACPECPKRFMRSDNLTQHIKTH (SEQ ID NO: 112);

[0115] ZFP17:

[0116] YKCPECGKSFSDPGHLVRHQRTH (SEQ ID NO: 113),

[0117] YKCPECGKSFSTSHSLTEHQRTH (SEQ ID NO: 114),

[0118] YECHDCGKSFRQSTHLTQHRRIH (SEQ ID NO: 115), and

[0119] YTCSDCGKAFRDKSCLNRHRRTH(SEQ ID NO: 116);

[0120] ZFP18:

[0121] YHCDWDGCGWKFARSDELTRHYRKH (SEQ ID NO: 117),

[0122] YKCPECGKSFSRADNLTEHQRTH (SEQ ID NO: 118),

[0123] YRCKYCDRSFSDSSNLQRHVRNIH (SEQ ID NO: 119), and

[0124] YKCPECGKSFSTSHSLTEHQRTH (SEQ ID NO: 120);

[0125] ZFP19:

[0126] YSCGICGKSFSDSSAKRRHCILH (SEQ ID NO: 121),

[0127] YKCPECGKSFSDPGHLVRHQRTH (SEQ ID NO: 122),

[0128] YKCPECGKSFSTSHSLTEHQRTH (SEQ ID NO: 123), and

[0129] YECHDCGKSFRQSTHLTQHRRIH (SEQ ID NO: 124);

[0130] ZFP20:

[0131] YECVQCGKGFTQSSNLITHQRVH (SEQ ID NO: 125),

[0132] FQCKTCQRKFSRSDHLKTHTRTH (SEQ ID NO: 126),

[0133] FQCRICMRNFSDSGNLRVHIRTH (SEQ ID NO: 127),

[0134] YTCSYCGKSFTQSNTLKQHTRIH (SEQ ID NO: 128), and

[0135] YECDHCGKSFSQSSHLNVHKRTH (SEQ ID NO: 129);

[0136] ZFP21:

[0137] YRCKYCDRSFSDSSNLQRHVRNIH (SEQ ID NO: 130),

[0138] YECVQCGKGFTQSSNLITHQRVH (SEQ ID NO: 131),

[0139] FQCKTCQRKFSRSDHLKTHTRTH (SEQ ID NO: 132),

[0140] FQCRICMRNFSDSGNLRVHIRTH (SEQ ID NO: 133), and

[0141] YTCSYCGKSFTQSNTLKQHTRIH (SEQ ID NO: 134);

[0142] ZFP22:

[0143] YTCSDCGKAFRDKSCLNRHRRTH (SEQ ID NO: 135),

[0144] YRCKYCDRSFSDSSNLQRHVRNIH (SEQ ID NO: 136),

[0145] YECVQCGKGFTQSSNLITHQRVH (SEQ ID NO: 137),

[0146] FQCKTCQRKFSRSDHLKTHTRTH (SEQ ID NO: 138), and

[0147] FQCRICMRNFSDSGNLRVHIRTH(SEQ ID NO: 139);

[0148] ZFP23:

[0149] FQCKTCQRKFSRSDHLKTHTRTH (SEQ ID NO: 140),

[0150] YTCSDCGKAFRDKSCLNRHRRTH (SEQ ID NO: 141),

[0151] YRCKYCDRSFSDSSNLQRHVRNIH (SEQ ID NO: 142),

[0152] YECVQCGKGFTQSSNLITHQRVH (SEQ ID NO: 143), and

[0153] FQCKTCQRKFSRSDHLKTHTRTH (SEQ ID NO: 144);

[0154] ZFP24:

[0155] YKCPECGKSFSTSHSLTEHQRTH (SEQ ID NO: 145),

[0156] YKCPECGKSFSTSHSLTEHQRTH (SEQ ID NO: 146),

[0157] YKCPDCGKSFSQSSSLIRHQRTH (SEQ ID NO: 147),

[0158] YKCGQCGKFYSQVSHLTRHQKIH (SEQ ID NO: 148), and

[0159] YKCPECGKSFSRADNLTEHQRTH (SEQ ID NO: 149);

[0160] ZFP25:

[0161] YKCPECGKSFSRADNLTEHQRTH (SEQ ID NO: 150),

[0162] YKCPECGKSFSSKKALTEHQRTH (SEQ ID NO: 151),

[0163] YKCPECGKSFSSKKALTEHQRTH (SEQ ID NO: 152),

[0164] YGCHLCGKAFSKSSNLRRHEMIH (SEQ ID NO: 153), and

[0165] FQCKTCQRKFSRSDHLKTHTRTH (SEQ ID NO: 154);

[0166] ZFP26:

[0167] YKCMECGKAFNRRSHLTRHQRIH (SEQ ID NO: 155),

[0168] YKCPECGKSFSTSHSLTEHQRTH (SEQ ID NO: 156),

[0169] YKCPECGKSFSTSHSLTEHQRTH (SEQ ID NO: 157),

[0170] YKCPDCGKSFSQSSSLIRHQRTH (SEQ ID NO: 158), and

[0171] YKCGQCGKFYSQVSHLTRHQKIH (SEQ ID NO: 159);

[0172] ZFP27:

[0173] YKCGQCGKFYSQVSHLTRHQKIH (SEQ ID NO: 160),

[0174] YKCPECGKSFSRADNLTEHQRTH (SEQ ID NO: 161),

[0175] YKCPECGKSFSSKKALTEHQRTH (SEQ ID NO: 162),

[0176] YKCPECGKSFSSKKALTEHQRTH (SEQ ID NO: 163), and

[0177] YGCHLCGKAFSKSSNLRRHEMIH (SEQ ID NO: 164);

[0178] ZFP28:

[0179] FQCRICMRNFSDSGNLRVHIRTH (SEQ ID NO: 165),

[0180] YKCMECGKAFNRRSHLTRHQRIH (SEQ ID NO: 166),

[0181] YKCPECGKSFSTSHSLTEHQRTH (SEQ ID NO: 167),

[0182] YKCPECGKSFSTSHSLTEHQRTH (SEQ ID NO: 168), and

[0183] YKCPDCGKSFSQSSSLIRHQRTH (SEQ ID NO: 169);

[0184] ZFP29:

[0185] YKCPECGKSFSSPADLTRHQRTH (SEQ ID NO: 170),

[0186] YKCGQCGKFYSQVSHLTRHQKIH (SEQ ID NO: 171),

[0187] YKCPECGKSFSRADNLTEHQRTH (SEQ ID NO: 172),

[0188] YKCPECGKSFSSKKALTEHQRTH (SEQ ID NO: 173), and

[0189] YKCPECGKSFSSKKALTEHQRTH (SEQ ID NO: 174);

[0190] ZFP30:

[0191] YTCSDCGKAFRDKSCLNRHRRTH (SEQ ID NO: 175),

[0192] FECKDCGKAFIQKSNLIRHQRTH (SEQ ID NO: 176),

[0193] YKCPECGKSFSTHLDLIRHQRTH (SEQ ID NO: 177),

[0194] YKCPDCGKSFSQSSSLIRHQRTH (SEQ ID NO: 178), and

[0195] YKCPECGKSFSTKNSLTEHQRTH (SEQ ID NO: 179);

[0196] ZFP31:

[0197] YECHDCGKSFRQSTHLTQHRRIH (SEQ ID NO: 180),

[0198] YTCSDCGKAFRDKSCLNRHRRTH (SEQ ID NO: 181),

[0199] FECKDCGKAFIQKSNLIRHQRTH (SEQ ID NO: 182),

[0200] YKCPECGKSFSTHLDLIRHQRTH (SEQ ID NO: 183), and

[0201] YKCPDCGKSFSQSSSLIRHQRTH (SEQ ID NO: 184);

[0202] ZFP32:

[0203] YKCPECGKSFSTSHSLTEHQRTH (SEQ ID NO: 185),

[0204] YECHDCGKSFRQSTHLTQHRRIH (SEQ ID NO: 186),

[0205] YTCSDCGKAFRDKSCLNRHRRTH (SEQ ID NO: 187),

[0206] FECKDCGKAFIQKSNLIRHQRTH (SEQ ID NO: 188), and

[0207] YKCPECGKSFSTHLDLIRHQRTH (SEQ ID NO: 189);

[0208] ZFP33:

[0209] YKCPECGKSFSDPGHLVRHQRTH (SEQ ID NO: 190),

[0210] YKCPECGKSFSTSHSLTEHQRTH (SEQ ID NO: 191),

[0211] YECHDCGKSFRQSTHLTQHRRIH (SEQ ID NO: 192),

[0212] YTCSDCGKAFRDKSCLNRHRRTH (SEQ ID NO: 193), and

[0213] FECKDCGKAFIQKSNLIRHQRTH(SEQ ID NO: 194);

[0214] ZFP34:

[0215] YHCDWDGCGWKFARSDELTRHYRKH (SEQ ID NO: 195),

[0216] YKCPECGKSFSRADNLTEHQRTH (SEQ ID NO: 196),

[0217] YRCKYCDRSFSDSSNLQRHVRNIH (SEQ ID NO: 197),

[0218] YKCPECGKSFSTSHSLTEHQRTH (SEQ ID NO: 198), and

[0219] FACPECPKRFMRSDNLTQHIKTH (SEQ ID NO: 199);

[0220] ZFP35:

[0221] YSCGICGKSFSDSSAKRRHCILH (SEQ ID NO: 200),

[0222] YKCPECGKSFSDPGHLVRHQRTH (SEQ ID NO: 201),

[0223] YKCPECGKSFSTSHSLTEHQRTH (SEQ ID NO: 202),

[0224] YECHDCGKSFRQSTHLTQHRRIH (SEQ ID NO: 203), and

[0225] YTCSDCGKAFRDKSCLNRHRRTH(SEQ ID NO: 204);

[0226] ZFP36:

[0227] YRCKYCDRSFSDSSNLQRHVRNIH (SEQ ID NO: 205),

[0228] YECVQCGKGFTQSSNLITHQRVH (SEQ ID NO: 206),

[0229] FQCKTCQRKFSRSDHLKTHTRTH (SEQ ID NO: 207),

[0230] FQCRICMRNFSDSGNLRVHIRTH (SEQ ID NO: 208),

[0231] YTCSYCGKSFTQSNTLKQHTRIH (SEQ ID NO: 209), and

[0232] YECDHCGKSF SQSSHLNVHKRTH (SEQ ID NO: 210);

[0233] ZFP37:

[0234] YTCSDCGKAFRDKSCLNRHRRTH (SEQ ID NO: 211),

[0235] YRCKYCDRSFSDSSNLQRHVRNIH (SEQ ID NO: 212),

[0236] YECVQCGKGFTQSSNLITHQRVH (SEQ ID NO: 213),

[0237] FQCKTCQRKFSRSDHLKTHTRTH (SEQ ID NO: 214),

[0238] FQCRICMRNFSDSGNLRVHIRTH (SEQ ID NO: 215), and

[0239] YTCSYCGKSF TQSNTLKQHTRIH (SEQ ID NO: 216);

[0240] ZFP38:

[0241] FQCKTCQRKFSRSDHLKTHTRTH (SEQ ID NO: 217),

[0242] YTCSDCGKAFRDKSCLNRHRRTH (SEQ ID NO: 218),

[0243] YRCKYCDRSFSDSSNLQRHVRNIH (SEQ ID NO: 219),

[0244] YECVQCGKGFTQSSNLITHQRVH (SEQ ID NO: 220),

[0245] FQCKTCQRKFSRSDHLKTHTRTH (SEQ ID NO: 221), and

[0246] FQCRICMRNFSDSGNLRVHIRTH(SEQ ID NO: 222);

[0247] ZFP39:

[0248] YKCMECGKAFNRRSHLTRHQRIH (SEQ ID NO: 223),

[0249] YKCPECGKSFSTSHSLTEHQRTH (SEQ ID NO: 224),

[0250] YKCPECGKSFSTSHSLTEHQRTH (SEQ ID NO: 225),

[0251] YKCPDCGKSFSQSSSLIRHQRTH (SEQ ID NO: 226),

[0252] YKCGQCGKFYSQVSHLTRHQKIH (SEQ ID NO: 227), and

[0253] YKCPECGKSFSRADNLTEHQRTH (SEQ ID NO: 228);

[0254] ZFP40:

[0255] YKCGQCGKFYSQVSHLTRHQKIH (SEQ ID NO: 229),

[0256] YKCPECGKSFSRADNLTEHQRTH (SEQ ID NO: 230),

[0257] YKCPECGKSFSSKKALTEHQRTH (SEQ ID NO: 231),

[0258] YKCPECGKSFSSKKALTEHQRTH (SEQ ID NO: 232),

[0259] YGCHLCGKAFSKSSNLRRHEMIH (SEQ ID NO: 233), and

[0260] FQCKTCQRKFSRSDHLKTHTRTH (SEQ ID NO: 234);

[0261] ZFP41:

[0262] FQCRICMRNFSDSGNLRVHIRTH (SEQ ID NO: 235),

[0263] YKCMECGKAFNRRSHLTRHQRIH (SEQ ID NO: 236),

[0264] YKCPECGKSFSTSHSLTEHQRTH (SEQ ID NO: 237),

[0265] YKCPECGKSFSTSHSLTEHQRTH (SEQ ID NO: 238),

[0266] YKCPDCGKSFSQSSSLIRHQRTH (SEQ ID NO: 239), and

[0267] YKCGQCGKFYSQVSHLTRHQKIH (SEQ ID NO: 240);

[0268] ZFP42:

[0269] YKCPECGKSFSSPADLTRHQRTH (SEQ ID NO: 241),

[0270] YKCGQCGKFYSQVSHLTRHQKIH (SEQ ID NO: 242),

[0271] YKCPECGKSFSRADNLTEHQRTH (SEQ ID NO: 243),

[0272] YKCPECGKSFSSKKALTEHQRTH (SEQ ID NO: 244),

[0273] YKCPECGKSFSSKKALTEHQRTH (SEQ ID NO: 245), and

[0274] YGCHLCGKAFSKSSNLRRHEMIH (SEQ ID NO: 246);

[0275] ZFP43:

[0276] YECHDCGKSFRQSTHLTQHRRIH (SEQ ID NO: 247),

[0277] YTCSDCGKAFRDKSCLNRHRRTH (SEQ ID NO: 248),

[0278] FECKDCGKAFIQKSNLIRHQRTH (SEQ ID NO: 249),

[0279] YKCPECGKSFSTHLDLIRHQRTH (SEQ ID NO: 250),

[0280] YKCPDCGKSFSQSSSLIRHQRTH (SEQ ID NO: 251), and

[0281] YKCPECGKSFSTKNSLTEHQRTH (SEQ ID NO: 252);

[0282] ZFP44:

[0283] YKCPECGKSFSTSHSLTEHQRTH (SEQ ID NO: 253),

[0284] YECHDCGKSFRQSTHLTQHRRIH (SEQ ID NO: 254),

[0285] YTCSDCGKAFRDKSCLNRHRRTH (SEQ ID NO: 255),

[0286] FECKDCGKAFIQKSNLIRHQRTH (SEQ ID NO: 256),

[0287] YKCPECGKSFSTHLDLIRHQRTH (SEQ ID NO: 257), and

[0288] YKCPDCGKSFSQSSSLIRHQRTH (SEQ ID NO: 258);

[0289] ZFP45:

[0290] YKCPECGKSFSDPGHLVRHQRTH (SEQ ID NO: 259),

[0291] YKCPECGKSFSTSHSLTEHQRTH (SEQ ID NO: 260),

[0292] YECHDCGKSFRQSTHLTQHRRIH (SEQ ID NO: 261),

[0293] YTCSDCGKAFRDKSCLNRHRRTH (SEQ ID NO: 262),

[0294] FECKDCGKAFIQKSNLIRHQRTH (SEQ ID NO: 263), and

[0295] YKCPECGKSFSTHLDLIRHQRTH (SEQ ID NO: 264);

[0296] ZFP46:

[0297] YSCGICGKSFSDSSAKRRHCILH (SEQ ID NO: 265),

[0298] YKCPECGKSFSDPGHLVRHQRTH (SEQ ID NO: 266),

[0299] YKCPECGKSFSTSHSLTEHQRTH (SEQ ID NO: 267),

[0300] YECHDCGKSFRQSTHLTQHRRIH (SEQ ID NO: 268),

[0301] YTCSDCGKAFRDKSCLNRHRRTH (SEQ ID NO: 269), and

[0302] FECKDCGKAFIQKSNLIRHQRTH (SEQ ID NO: 270);

[0303] sZFP1:

[0304] YKCGQCGKFYSQVSHLTRHQKIH (SEQ ID NO: 271),

[0305] YKCPECGKSFSRADNLTEHQRTH (SEQ ID NO: 272),

[0306] YKCPECGKSFSSKKALTEHQRTH (SEQ ID NO: 273),

[0307] YKCPDCGKSFSQSSSLIRHQRTH (SEQ ID NO: 274),

[0308] YKCGQCGKFYSQVSHLTRHQKIH (SEQ ID NO: 275), and

[0309] YKCPECGKSFSRADNLTEHQRTH (SEQ ID NO: 276);

[0310] sZFP3:

[0311] YKCPECGKSFSSPADLTRHQRTH (SEQ ID NO: 277),

[0312] YKCGQCGKFYSQVSHLTRHQKIH (SEQ ID NO: 278),

[0313] YKCPECGKSFSRADNLTEHQRTH (SEQ ID NO: 279),

[0314] YKCPECGKSFSTSHSLTEHQRTH (SEQ ID NO: 280),

[0315] YKCPDCGKSFSQSSSLIRHQRTH (SEQ ID NO: 281), and

[0316] YKCGQCGKFYSQVSHLTRHQKIH (SEQ ID NO: 282);

[0317] sZFP5:

[0318] YKCPECGKSFSTHLDLIRHQRTH (SEQ ID NO: 283),

[0319] YKCPDCGKSFSQSSSLIRHQRTH (SEQ ID NO: 284),

[0320] YKCPECGKSFSTKNSLTEHQRTH (SEQ ID NO: 285),

[0321] YECNYCGKTFSVSSTLIRHQRIH (SEQ ID NO: 286),

[0322] YRCEECGKAFRWPSNLTRHKRIH (SEQ ID NO: 287), and

[0323] YRCEECGKAFRWPSNLTRHKRIH (SEQ ID NO: 288);

[0324] sZFP7:

[0325] YRCKYCDRSFSDSSNLQRHVRNIH (SEQ ID NO: 289),

[0326] YKCPECGKSFSTSHSLTEHQRTH (SEQ ID NO: 290),

[0327] FACPECPKRFMRSDNLTQHIKTH (SEQ ID NO: 291),

[0328] YKCPECGKSFSTHLDLIRHQRTH (SEQ ID NO: 292),

[0329] YKCPDCGKSFSQSSSLIRHQRTH (SEQ ID NO: 293), and

[0330] YKCPECGKSFSTKNSLTEHQRTH (SEQ ID NO: 294);

[0331] sZFP9:

[0332] YKCPECGKSFSRADNLTEHQRTH (SEQ ID NO: 295),

[0333] YRCKYCDRSFSDSSNLQRHVRNIH (SEQ ID NO: 296),

[0334] YKCPECGKSFSTSHSLTEHQRTH (SEQ ID NO: 297),

[0335] FECKDCGKAFIQKSNLIRHQRTH (SEQ ID NO: 298),

[0336] YKCPECGKSFSTHLDLIRHQRTH (SEQ ID NO: 299), and

[0337] YKCPDCGKSFSQSSSLIRHQRTH (SEQ ID NO: 300);

[0338] sZFP12:

[0339] YHCDWDGCGWKFARSDELTRHYRKH (SEQ ID NO: 301),

[0340] YKCPECGKSFSRADNLTEHQRTH (SEQ ID NO: 302),

[0341] YRCKYCDRSFSDSSNLQRHVRNIH (SEQ ID NO: 303),

[0342] YTCSDCGKAFRDKSCLNRHRRTH (SEQ ID NO: 304),

[0343] FECKDCGKAFIQKSNLIRHQRTH (SEQ ID NO: 305), and

[0344] YKCPECGKSFSTHLDLIRHQRTH (SEQ ID NO: 306);

[0345] The ZFP according to the present invention may comprise three or more zinc finger motif proteins linked to each other via a linker. The linker may be a peptide linker comprising 2 to 50 amino acid residues. The amino acid residues and / or length of the linker may vary as long as the function of the ZFP is not affected.

[0346] The ZFP according to the present invention may include, in addition to three or more zinc finger motif proteins and a linker, a peptide comprising 2 to 50 amino acid residues at the N-terminus and / or C-terminus. For example, a peptide or a portion thereof constituting a domain located at the N-terminus of a zinc finger existing in nature may be used.

[0347] The ZFP according to the present invention may include a nuclear localization signal (NLS) sequence for nuclear transport. The NLS sequence may be any signal sequence that confers the ability to translocate into the nucleus, and any known NLS sequence or variant sequence thereof with NLS function may be used. In addition, the ZFP according to the present invention may include additional sequences for biotechnology techniques, such as tags.

[0348] The ZFP according to the present invention may be selected from the group consisting of, for example, the following amino acid sequences (in the amino acid sequences below, the portions indicated in bold represent zinc finger motifs).

[0349] ZFP1

[0350] MRKYPNRPSKTPPHERPFECKDCGKAFIQKSNLIRHQRTHTGEKPYKCPECGKSFSTKNSLTEHQRTHTGEKPYTCSDCGKAFRDKSCLNRHRRTHTGEKPYKCPECGKSFSTSHSLTEHQRTHTGEKPAAKRVKLDDYKDDDDK(서열번호 307)

[0351] ZFP2

[0352] MRKYPNRPSKTPPHERPYECVQCGKGFTQSSNLITHQRVHTGEKPFQCKTCQRKFSRSDHLKTHTRTHTGEKPFQCRICMRNFSDSGNLRVHIRTHTGEKPYTCSYCGKSFTQSNTLKQHTRIHTGEKPAAKRVKLDDYKDDDDK(서열번호 308)

[0353] ZFP3

[0354] MRKYPNRPSKTPPHERPYTCSDCGKAFRDKSCLNRHRRTHTGEKPYRCKYCDRSFSDSSNLQRHVRNIHTGEKPYECVQCGKGFTQSSNLITHQRVHTGEKPFQCKTCQRKFSRSDHLKTHTRTHTGEKPAAKRVKLDDYKDDDDK(서열번호 309)

[0355] ZFP4

[0356] MRKYPNRPSKTPPHERPFQCKTCQRKFSRSDHLKTHTRTHTGEKPYTCSDCGKAFRDKSCLNRHRRTHTGEKPYRCKYCDRSFSDSSNLQRHVRNIHTGEKPYECVQCGKGFTQSSNLITHQRVHTGEKPAAKRVKLDDYKDDDDK(서열번호 310)

[0357] ZFP5

[0358] MRKYPNRPSKTPPHERPYKCPECGKSFSTSHSLTEHQRTHTGEKPYKCPDCGKSFSQSSSLIRHQRTHTGEKPYKCGQCGKFYSQVSHLTRHQKIHTGEKPYKCPECGKSFSRADNLTEHQRTHTGEKPAAKRVKLDDYKDDDDK(서열번호 311)

[0359] ZFP6

[0360] MRKYPNRPSKTPPHERPYKCPECGKSFSSKKALTEHQRTHTGEKPYKCPECGKSFSSKKALTEHQRTHTGEKPYGCHLCGKAFSKSSNLRRHEMIHTGEKPFQCKTCQRKFSRSDHLKTHTRTHTGEKPAAKRVKLDDYKDDDDK(서열번호 312)

[0361] ZFP7

[0362] MRKYPNRPSKTPPHERPYKCMECGKAFNRRSHLTRHQRIHTGEKPYKCPECGKSFSTSHSLTEHQRTHTGEKPYKCPECGKSFSTSHSLTEHQRTHTGEKPYKCPDCGKSFSQSSSLIRHQRTHTGEKPAAKRVKLDDYKDDDDK(서열번호 313)

[0363] ZFP8

[0364] MRKYPNRPSKTPPHERPYKCGQCGKFYSQVSHLTRHQKIHTGEKPYKCPECGKSFSRADNLTEHQRTHTGEKPYKCPECGKSFSSKKALTEHQRTHTGEKPYKCPECGKSFSSKKALTEHQRTHTGEKPAAKRVKLDDYKDDDDK(서열번호 314)

[0365] ZFP9

[0366] MRKYPNRPSKTPPHERPFQCRICMRNFSDSGNLRVHIRTHTGEKPYKCMECGKAFNRRSHLTRHQRIHTGEKPYKCPECGKSFSTSHSLTEHQRTHTGEKPYKCPECGKSFSTSHSLTEHQRTHTGEKPAAKRVKLDDYKDDDDK(서열번호 315)

[0367] ZFP10

[0368] MRKYPNRPSKTPPHERPYKCPECGKSFSSPADLTRHQRTHTGEKPYKCGQCGKFYSQVSHLTRHQKIHTGEKPYKCPECGKSFSRADNLTEHQRTHTGEKPYKCPECGKSFSSKKALTEHQRTHTGEKPAAKRVKLDDYKDDDDK(서열번호 316)

[0369] ZFP11

[0370] MRKYPNRPSKTPPHERPYECNYCGKTFSVSSTLIRHQRIHTGEKPYRCEECGKAFRWPSNLTRHKRIHTGEKPYRCEECGKAFRWPSNLTRHKRIHTGEKPYKCPECGKSFSTKNSLTEHQRTHTGEKPAAKRVKLDDYKDDDDK(서열번호 317)

[0371] ZFP12

[0372] MRKYPNRPSKTPPHERPFECKDCGKAFIQKSNLIRHQRTHTGEKPYKCPECGKSFSTHLDLIRHQRTHTGEKPYKCPDCGKSFSQSSSLIRHQRTHTGEKPYKCPECGKSFSTKNSLTEHQRTHTGEKPAAKRVKLDDYKDDDDK(서열번호 318)

[0373] ZFP13

[0374] MRKYPNRPSKTPPHERPYTCSDCGKAFRDKSCLNRHRRTHTGEKPFECKDCGKAFIQKSNLIRHQRTHTGEKPYKCPECGKSFSTHLDLIRHQRTHTGEKPYKCPDCGKSFSQSSSLIRHQRTHTGEKPAAKRVKLDDYKDDDDK(서열번호 319)

[0375] ZFP14

[0376] MRKYPNRPSKTPPHERPYECHDCGKSFRQSTHLTQHRRIHTGEKPYTCSDCGKAFRDKSCLNRHRRTHTGEKPFECKDCGKAFIQKSNLIRHQRTHTGEKPYKCPECGKSFSTHLDLIRHQRTHTGEKPAAKRVKLDDYKDDDDK(서열번호 320)

[0377] ZFP15

[0378] MRKYPNRPSKTPPHERPYKCPECGKSFSTSHSLTEHQRTHTGEKPYECHDCGKSFRQSTHLTQHRRIHTGEKPYTCSDCGKAFRDKSCLNRHRRTHTGEKPFECKDCGKAFIQKSNLIRHQRTHTGEKPAAKRVKLDDYKDDDDK(서열번호 321)

[0379] ZFP16

[0380] MRKYPNRPSKTPPHERPYKCPECGKSFSRADNLTEHQRTHTGEKPYRCKYCDRSFSDSSNLQRHVRNIHTGEKPYKCPECGKSFSTSHSLTEHQRTHTGEKPFACPECPKRFMRSDNLTQHIKTHTGEKPAAKRVKLDDYKDDDDK(서열번호 322)

[0381] ZFP17

[0382] MRKYPNRPSKTPPHERPYKCPECGKSFSDPGHLVRHQRTHTGEKPYKCPECGKSFSTSHSLTEHQRTHTGEKPYECHDCGKSFRQSTHLTQHRRIHTGEKPYTCSDCGKAFRDKSCLNRHRRTHTGEKPAAKRVKLDDYKDDDDK(서열번호 323)

[0383] ZFP18

[0384] MRKYPNRPSKTPPHERPYHCDWDGCGWKFARSDELTRHYRKHTGEKPYKCPECGKSFSRADNLTEHQRTHTGEKPYRCKYCDRSFSDSSNLQRHVRNIHTGEKPYKCPECGKSFSTSHSLTEHQRTHTGEKPAAKRVKLDDYKDDDDK(서열번호 324)

[0385] ZFP19

[0386] MRKYPNRPSKTPPHERPYSCGICGKSFSDSSAKRRHCILHTGEKPYKCPECGKSFSDPGHLVRHQRTHTGEKPYKCPECGKSFSTSHSLTEHQRTHTGEKPYECHDCGKSFRQSTHLTQHRRIHTGEKPAAKRVKLDDYKDDDDK(서열번호 325)

[0387] ZFP20

[0388] MRKYPNRPSKTPPHERPYECVQCGKGFTQSSNLITHQRVHTGEKPFQCKTCQRKFSRSDHLKTHTRTHTGEKPFQCRICMRNFSDSGNLRVHIRTHTGEKPYTCSYCGKSFTQSNTLKQHTRIHTGEKPYECDHCGKSFSQSSHLNVHKRTHTGEKPAAKRVKLDDYKDDDDK(서열번호 326)

[0389] ZFP21

[0390] MRKYPNRPSKTPPHERPYRCKYCDRSFSDSSNLQRHVRNIHTGEKPYECVQCGKGFTQSSNLITHQRVHTGEKPFQCKTCQRKFSRSDHLKTHTRTHTGEKPFQCRICMRNFSDSGNLRVHIRTHTGEKPYTCSYCGKSFTQSNTLKQHTRIHTGEKPAAKRVKLDDYKDDDDK(서열번호 327)

[0391] ZFP22

[0392] MRKYPNRPSKTPPHERPYTCSDCGKAFRDKSCLNRHRRTHTGEKPYRCKYCDRSFSDSSNLQRHVRNIHTGEKPYECVQCGKGFTQSSNLITHQRVHTGEKPFQCKTCQRKFSRSDHLKTHTRTHTGEKPFQCRICMRNFSDSGNLRVHIRTHTGEKPAAKRVKLDDYKDDDDK(서열번호 328)

[0393] ZFP23

[0394] MRKYPNRPSKTPPHERPFQCKTCQRKFSRSDHLKTHTRTHTGEKPYTCSDCGKAFRDKSCLNRHRRTHTGEKPYRCKYCDRSFSDSSNLQRHVRNIHTGEKPYECVQCGKGFTQSSNLITHQRVHTGEKPFQCKTCQRKFSRSDHLKTHTRTHTGEKPAAKRVKLDDYKDDDDK(서열번호 329)

[0395] ZFP24

[0396] MRKYPNRPSKTPPHERPYKCPECGKSFSTSHSLTEHQRTHTGEKPYKCPECGKSFSTSHSLTEHQRTHTGEKPYKCPDCGKSFSQSSSLIRHQRTHTGEKPYKCGQCGKFYSQVSHLTRHQKIHTGEKPYKCPECGKSFSRADNLTEHQRTHTGEKPAAKRVKLDDYKDDDDK(서열번호 330)

[0397] ZFP25

[0398] MRKYPNRPSKTPPHERPYKCPECGKSFSRADNLTEHQRTHTGEKPYKCPECGKSFSSKKALTEHQRTHTGEKPYKCPECGKSFSSKKALTEHQRTHTGEKPYGCHLCGKAFSKSSNLRRHEMIHTGEKPFQCKTCQRKFSRSDHLKTHTRTHTGEKPAAKRVKLDDYKDDDDK(서열번호 331)

[0399] ZFP26

[0400] MRKYPNRPSKTPPHERPYKCMECGKAFNRRSHLTRHQRIHTGEKPYKCPECGKSFSTSHSLTEHQRTHTGEKPYKCPECGKSFSTSHSLTEHQRTHTGEKPYKCPDCGKSFSQSSSLIRHQRTHTGEKPYKCGQCGKFYSQVSHLTRHQKIHTGEKPAAKRVKLDDYKDDDDK(서열번호 332)

[0401] ZFP27

[0402] MRKYPNRPSKTPPHERPYKCGQCGKFYSQVSHLTRHQKIHTGEKPYKCPECGKSFSRADNLTEHQRTHTGEKPYKCPECGKSFSSKKALTEHQRTHTGEKPYKCPECGKSFSSKKALTEHQRTHTGEKPYGCHLCGKAFSKSSNLRRHEMIHTGEKPAAKRVKLDDYKDDDDK(서열번호 333)

[0403] ZFP28

[0404] MRKYPNRPSKTPPHERPFQCRICMRNFSDSGNLRVHIRTHTGEKPYKCMECGKAFNRRSHLTRHQRIHTGEKPYKCPECGKSFSTSHSLTEHQRTHTGEKPYKCPECGKSFSTSHSLTEHQRTHTGEKPYKCPDCGKSFSQSSSLIRHQRTHTGEKPAAKRVKLDDYKDDDDK(서열번호 334)

[0405] ZFP29

[0406] MRKYPNRPSKTPPHERPYKCPECGKSFSSPADLTRHQRTHTGEKPYKCGQCGKFYSQVSHLTRHQKIHTGEKPYKCPECGKSFSRADNLTEHQRTHTGEKPYKCPECGKSFSSKKALTEHQRTHTGEKPYKCPECGKSFSSKKALTEHQRTHTGEKPAAKRVKLDDYKDDDDK(서열번호 335)

[0407] ZFP30

[0408] MRKYPNRPSKTPPHERPYTCSDCGKAFRDKSCLNRHRRTHTGEKPFECKDCGKAFIQKSNLIRHQRTHTGEKPYKCPECGKSFSTHLDLIRHQRTHTGEKPYKCPDCGKSFSQSSSLIRHQRTHTGEKPYKCPECGKSFSTKNSLTEHQRTHTGEKPAAKRVKLDDYKDDDDK(서열번호 336)

[0409] ZFP31

[0410] MRKYPNRPSKTPPHERPYECHDCGKSFRQSTHLTQHRRIHTGEKPYTCSDCGKAFRDKSCLNRHRRTHTGEKPFECKDCGKAFIQKSNLIRHQRTHTGEKPYKCPECGKSFSTHLDLIRHQRTHTGEKPYKCPDCGKSFSQSSSLIRHQRTHTGEKPAAKRVKLDDYKDDDDK(서열번호 337)

[0411] ZFP32

[0412] MRKYPNRPSKTPPHERPYKCPECGKSFSTSHSLTEHQRTHTGEKPYECHDCGKSFRQSTHLTQHRRIHTGEKPYTCSDCGKAFRDKSCLNRHRRTHTGEKPFECKDCGKAFIQKSNLIRHQRTHTGEKPYKCPECGKSFSTHLDLIRHQRTHTGEKPAAKRVKLDDYKDDDDK(서열번호 338)

[0413] ZFP33

[0414] MRKYPNRPSKTPPHERPYKCPECGKSFSDPGHLVRHQRTHTGEKPYKCPECGKSFSTSHSLTEHQRTHTGEKPYECHDCGKSFRQSTHLTQHRRIHTGEKPYTCSDCGKAFRDKSCLNRHRRTHTGEKPFECKDCGKAFIQKSNLIRHQRTHTGEKPAAKRVKLDDYKDDDDK(서열번호 339)

[0415] ZFP34

[0416] MRKYPNRPSKTPPHERPYHCDWDGCGWKFARSDELTRHYRKHTGEKPYKCPECGKSFSRADNLTEHQRTHTGEKPYRCKYCDRSFSDSSNLQRHVRNIHTGEKPYKCPECGKSFSTSHSLTEHQRTHTGEKPFACPECPKRFMRSDNLTQHIKTHTGEKPAAKRVKLDDYKDDDDK(서열번호 340)

[0417] ZFP35

[0418] MRKYPNRPSKTPPHERPYSCGICGKSFSDSSAKRRHCILHTGEKPYKCPECGKSFSDPGHLVRHQRTHTGEKPYKCPECGKSFSTSHSLTEHQRTHTGEKPYECHDCGKSFRQSTHLTQHRRIHTGEKPYTCSDCGKAFRDKSCLNRHRRTHTGEKPAAKRVKLDDYKDDDDK(서열번호 341)

[0419] ZFP36

[0420] MRKYPNRPSKTPPHERPYRCKYCDRSFSDSSNLQRHVRNIHTGEKPYECVQCGKGFTQSSNLITHQRVHTGEKPFQCKTCQRKFSRSDHLKTHTRTHTGEKPFQCRICMRNFSDSGNLRVHIRTHTGEKPYTCSYCGKSFTQSNTLKQHTRIHTGEKPYECDHCGKSFSQSSHLNVHKRTHTGEKPAAKRVKLDDYKDDDDK(서열번호 342)

[0421] ZFP37

[0422] MRKYPNRPSKTPPHERPYTCSDCGKAFRDKSCLNRHRRTHTGEKPYRCKYCDRSFSDSSNLQRHVRNIHTGEKPYECVQCGKGFTQSSNLITHQRVHTGEKPFQCKTCQRKFSRSDHLKTHTRTHTGEKPFQCRICMRNFSDSGNLRVHIRTHTGEKPYTCSYCGKSFTQSNTLKQHTRIHTGEKPAAKRVKLDDYKDDDDK(서열번호 343)

[0423] ZFP38

[0424] MRKYPNRPSKTPPHERPFQCKTCQRKFSRSDHLKTHTRTHTGEKPYTCSDCGKAFRDKSCLNRHRRTHTGEKPYRCKYCDRSFSDSSNLQRHVRNIHTGEKPYECVQCGKGFTQSSNLITHQRVHTGEKPFQCKTCQRKFSRSDHLKTHTRTHTGEKPFQCRICMRNFSDSGNLRVHIRTHTGEKPAAKRVKLDDYKDDDDK(서열번호 344)

[0425] ZFP39

[0426] MRKYPNRPSKTPPHERPYKCMECGKAFNRRSHLTRHQRIHTGEKPYKCPECGKSFSTSHSLTEHQRTHTGEKPYKCPECGKSFSTSHSLTEHQRTHTGEKPYKCPDCGKSFSQSSSLIRHQRTHTGEKPYKCGQCGKFYSQVSHLTRHQKIHTGEKPYKCPECGKSFSRADNLTEHQRTHTGEKPAAKRVKLDDYKDDDDK(서열번호 345)

[0427] ZFP40

[0428] MRKYPNRPSKTPPHERPYKCGQCGKFYSQVSHLTRHQKIHTGEKPYKCPECGKSFSRADNLTEHQRTHTGEKPYKCPECGKSFSSKKALTEHQRTHTGEKPYKCPECGKSFSSKKALTEHQRTHTGEKPYGCHLCGKAFSKSSNLRRHEMIHTGEKPFQCKTCQRKFSRSDHLKTHTRTHTGEKPAAKRVKLDDYKDDDDK(서열번호 346)

[0429] ZFP41

[0430] MRKYPNRPSKTPPHERPFQCRICMRNFSDSGNLRVHIRTHTGEKPYKCMECGKAFNRRSHLTRHQRIHTGEKPYKCPECGKSFSTSHSLTEHQRTHTGEKPYKCPECGKSFSTSHSLTEHQRTHTGEKPYKCPDCGKSFSQSSSLIRHQRTHTGEKPYKCGQCGKFYSQVSHLTRHQKIHTGEKPAAKRVKLDDYKDDDDK(서열번호 347)

[0431] ZFP42

[0432] MRKYPNRPSKTPPHERPYKCPECGKSFSSPADLTRHQRTHTGEKPYKCGQCGKFYSQVSHLTRHQKIHTGEKPYKCPECGKSFSRADNLTEHQRTHTGEKPYKCPECGKSFSSKKALTEHQRTHTGEKPYKCPECGKSFSSKKALTEHQRTHTGEKPYGCHLCGKAFSKSSNLRRHEMIHTGEKPAAKRVKLDDYKDDDDK(서열번호 348)

[0433] ZFP43

[0434] MRKYPNRPSKTPPHERPYECHDCGKSFRQSTHLTQHRRIHTGEKPYTCSDCGKAFRDKSCLNRHRRTHTGEKPFECKDCGKAFIQKSNLIRHQRTHTGEKPYKCPECGKSFSTHLDLIRHQRTHTGEKPYKCPDCGKSFSQSSSLIRHQRTHTGEKPYKCPECGKSFSTKNSLTEHQRTHTGEKPAAKRVKLDDYKDDDDK(서열번호 349)

[0435] ZFP44

[0436] MRKYPNRPSKTPPHERPYKCPECGKSFSTSHSLTEHQRTHTGEKPYECHDCGKSFRQSTHLTQHRRIHTGEKPYTCSDCGKAFRDKSCLNRHRRTHTGEKPFECKDCGKAFIQKSNLIRHQRTHTGEKPYKCPECGKSFSTHLDLIRHQRTHTGEKPYKCPDCGKSFSQSSSLIRHQRTHTGEKPAAKRVKLDDYKDDDDK(서열번호 350)

[0437] ZFP45

[0438] MRKYPNRPSKTPPHERPYKCPECGKSFSDPGHLVRHQRTHTGEKPYKCPECGKSFSTSHSLTEHQRTHTGEKPYECHDCGKSFRQSTHLTQHRRIHTGEKPYTCSDCGKAFRDKSCLNRHRRTHTGEKPFECKDCGKAFIQKSNLIRHQRTHTGEKPYKCPECGKSFSTHLDLIRHQRTHTGEKPAAKRVKLDDYKDDDDK(서열번호 351)

[0439] ZFP46

[0440] MRKYPNRPSKTPPHERPYSCGICGKSFSDSSAKRRHCILHTGEKPYKCPECGKSFSDPGHLVRHQRTHTGEKPYKCPECGKSFSTSHSLTEHQRTHTGEKPYECHDCGKSFRQSTHLTQHRRIHTGEKPYTCSDCGKAFRDKSCLNRHRRTHTGEKPFECKDCGKAFIQKSNLIRHQRTHTGEKPAAKRVKLDDYKDDDDK(서열번호 352)

[0441] sZFP1

[0442] MRKYPNRPSKTPPHERPYKCGQCGKFYSQVSHLTRHQKIHTGEKPYKCPECGKSFSRADNLTEHQRTHTGEKPYKCPECGKSFSSKKALTEHQRTHLRQKDGERPYKCPDCGKSFSQSSSLIRHQRTHTGEKPYKCGQCGKFYSQVSHLTRHQKIHTGEKPYKCPECGKSFSRADNLTEHQRTHTGEKPAAKRVKLDDYKDDDDK(서열번호 353)

[0443] sZFPs3

[0444] MRKYPNRPSKTPPHERPYKCPECGKSFSSPADLTRHQRTHTGEKPYKCGQCGKFYSQVSHLTRHQKIHTGEKPYKCPECGKSFSRADNLTEHQRTHLRQKDGERPYKCPECGKSFSTSHSLTEHQRTHTGEKPYKCPDCGKSFSQSSSLIRHQRTHTGEKPYKCGQCGKFYSQVSHLTRHQKIHTGEKPAAKRVKLDDYKDDDDK(서열번호 354)

[0445] sZFP5

[0446] MRKYPNRPSKTPPHERPYKCPECGKSFSTHLDLIRHQRTHTGEKPYKCPDCGKSFSQSSSLIRHQRTHTGEKPYKCPECGKSFSTKNSLTEHQRTHLRQKDGERPYECNYCGKTFSVSSTLIRHQRIHTGEKPYRCEECGKAFRWPSNLTRHKRIHTGEKPYRCEECGKAFRWPSNLTRHKRIHTGEKPAAKRVKLDDYKDDDDK(서열번호 355)

[0447] sZFP7

[0448] MRKYPNRPSKTPPHERPYRCKYCDRSFSDSSNLQRHVRNIHTGEKPYKCPECGKSFSTSHSLTEHQRTHTGEKPFACPECPKRFMRSDNLTQHIKTHLRQKDGER PYKCPECGKSFSTHLDLIRHQRTHTGEKPYKCPDCGKSFSQSSSLIRHQRTHTGEKPYKCPECGKSFSTKNSLTEHQRTHTGEKPAAKRVKLDDYKDDDDK (SEQ ID NO: 356)

[0449] sZFP9

[0450] MRKYPNRPSKTPPHERPYKCPECGKSFSRADNLTEHQRTHTGEKPYRCKYCDRSFSDSSNLQRHVRNIHTGEKPYKCPECGKSFSTSHSLTEHQRTHLRQKDGER PFECKDCGKAFIQKSNLIRHQRTHTGEKPYKCPECGKSFSTHLDLIRHQRTHTGEKPYKCPDCGKSFSQSSSLIRHQRTHTGEKPAAKRVKLDDYKDDDDK (SEQ ID NO: 357)

[0451] sZFP12

[0452] MRKYPNRPSKTPPHERPYHCDWDGCGWKFARSDELTRHYRKHTGEKPYKCPECGKSFSRADNLTEHQRTHTGEKPYRCKYCDRSFSDSSNLQRHVRNIHLRQKDGE RPYTCSDCGKAFRDKSCLNRHRRTHTGEKPFECKDCGKAFIQKSNLIRHQRTHTGEKPYKCPECGKSFSTHLDLIRHQRTHTGEKPAAKRVKLDDYKDDDDK (SEQ ID NO: 358)

[0453] ZFPs that can be used in the present invention can be used with or without a transcriptional repression domain. ZFPs that can be used in the present invention can effectively suppress the expression of the PMP22 protein even without a transcriptional repression domain.

[0454] The ZFP according to the present invention may reduce the expression of the PMP22 protein driven by the P1 promoter in Schwann cells of a patient with a disease related to Schwann cells by 20% or more, 30% or more, or 40% or more compared to a normal person. For example, the ZFP according to the present invention may reduce the expression of the PMP22 protein driven by the P1 promoter in Schwann cells of a patient with a disease related to Schwann cells by 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or 80% compared to a normal person. The disease related to Schwann cells may be CMT. The expression of the PMP22 protein may be achieved using techniques widely known in the field of biotechnology, such as a reporter system.

[0455] The present invention also provides an isolated polynucleotide encoding a CMT as described herein.

[0456] The polynucleotide according to the present invention may include a Schwann cell-specific promoter operably linked to the polynucleotide. The Schwann cell-specific promoter may be, but is not limited to, the MPZ promoter, the MBP promoter, the PMP22 promoter, or the MAG promoter.

[0457] As used herein, the term "operably linked" or similar expressions refer to the connection of a specific component with another component in gene expression technology such that the specific component functions in the intended manner. For example, when a promoter sequence is said to be operably linked to a coding sequence, it means that the promoter is linked so as to affect the transcription and / or expression of the coding sequence within a cell. Furthermore, the term encompasses all meanings that can be recognized by a person skilled in the art and may be appropriately interpreted according to the context.

[0458] transcription regulatory sequences

[0459] Another aspect of the present invention relates to a novel transcriptional regulatory sequence for increased expression of a nucleic acid molecule encoding a gene to be expressed in Schwann cells or a protein to be expressed in Schwann cells.

[0460] As used herein, the term “transcriptional regulatory sequence” means any sequence capable of regulating the transcription of an operably linked functional gene (gene of interest), which may be a promoter or may include a promoter and an enhancer together.

[0461] As used herein, the term "enhancer" refers to a nucleic acid sequence located adjacent to a promoter and capable of increasing the level of transcription by binding to a transcription factor. In other words, it refers to a nucleic acid sequence that can increase the transcriptional activity of a promoter when it is present compared to when it is absent.

[0462] In this specification, the term "promoter" has the conventional meaning known in the art, and refers to a nucleic acid sequence that has the function of controlling the transcription of one or more genes, which is located upstream of the translation initiation codon of a gene and includes a binding site for DNA-dependent RNA polymerase, a transcription initiation point, a binding site for a protein associated with transcription initiation, etc.

[0463] Specifically, the present invention provides a transcriptional regulatory sequence or a polynucleotide comprising the same, which comprises a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or at least 100% sequence identity with a nucleotide sequence selected from the group consisting of SEQ ID NOs: 363 to 366. In one embodiment of the present invention, the transcriptional regulatory sequence may comprise a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or at least 100% sequence identity with a nucleotide sequence of SEQ ID NOs: 365 or 366.

[0464] The term "sequence identity" or "identity" refers to the number (%) of identical amino acid residues or nucleotides matching at the same position, as measured from the alignment of two polypeptide or polynucleotide sequences. Sequence identity is determined by comparing the sequences when aligned to maximize overlap while minimizing sequence gaps. In particular, sequence identity can be determined using any of a number of mathematical global or local alignment algorithms, depending on the length of the two sequences. For example, sequence identity can be determined in various ways within the skill of the art, such as using publicly available computer software available at http: / / blast.ncbi.nlm.nih.gov / or http: / / www.ebi.ac.uk / Tools / emboss / .

[0465] One or more enhancers that may be used together with or as part of a transcription regulatory sequence according to the present invention may each independently comprise a polynucleotide having at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or at least 100% sequence identity with a nucleotide sequence selected from the group consisting of SEQ ID NOs: 359 to 362.

[0466] Preferably, the transcriptional regulatory sequence according to the present invention comprises a promoter and an enhancer together, and such transcriptional regulatory sequence may comprise a nucleotide sequence having a sequence identity of at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or at least 100% with a nucleotide sequence selected from the group consisting of SEQ ID NOs: 367 to 374.

[0467] The transcriptional regulatory sequence described above is used in a form in which the target gene is operably linked. In this respect, the present invention provides a polynucleotide comprising a transcriptional regulatory sequence and a target gene, wherein the transcriptional regulatory sequence comprises a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or at least 100% sequence identity with a nucleotide sequence selected from the group consisting of SEQ ID NOs: 363 to 374, and wherein the transcriptional regulatory sequence and the target gene are operably linked.

[0468] The above target gene may preferably be a nucleic acid encoding a gene to be expressed in Schwann cells or a protein to be expressed in Schwann cells.

[0469] The target gene may preferably be a nucleic acid encoding a zinc finger protein (ZFP) that binds to the promoter of the PMP22 gene or a part thereof in a nervous system cell to reduce the expression of the PMP22 protein. The promoter is preferably a P1 promoter.

[0470] The zinc finger protein can bind to a nucleic acid sequence portion selected from the group consisting of SEQ ID NOs: 3 to 48 and the following sequences or a complementary sequence thereof.

[0471] 5'-CAGGGAGCA-3'

[0472] 5'-CACCAGGGA-3'

[0473] 5'-GGAGCACCA-3'

[0474] 5'-CAGGGAACA-3'

[0475] 5'-GGTGGTGCT-3'

[0476] 5'-CCTGTAACT-3'

[0477] 5'-CCTGTAACT-3'

[0478] 5'-AAGCCAGAC-3'

[0479] 5'-GTAACTGAA-3'

[0480] 5'-CCAGACCAG-3'

[0481] 5'-ACTGAAGCC-3'

[0482] 5'-GACCAGGCG-3'

[0483] Additionally, the zinc finger protein may comprise a set of amino acid sequences selected from the group consisting of (1) to (52).

[0484] (1) Sequence numbers 49 to 52;

[0485] (2) Sequence numbers 53 to 56;

[0486] (3) Sequence numbers 57 to 60;

[0487] (4) Sequence numbers 61 to 64;

[0488] (5) Sequence numbers 65 to 68;

[0489] (6) Sequence numbers 69 to 72;

[0490] (7) Sequence numbers 73 to 76;

[0491] (8) Sequence numbers 77 to 80;

[0492] (9) Sequence numbers 81 to 84;

[0493] (10) Sequence numbers 85 to 88;

[0494] (11) Sequence numbers 89 to 92;

[0495] (12) Sequence numbers 93 to 96;

[0496] (13) Sequence numbers 97 to 100;

[0497] (14) Sequence numbers 101 to 104;

[0498] (15) Sequence numbers 105 to 108;

[0499] (16) Sequence numbers 109 to 112;

[0500] (17) Sequence numbers 113 to 116;

[0501] (18) Sequence numbers 117 to 120;

[0502] (19) Sequence numbers 121 to 124;

[0503] (20) Sequence numbers 125 to 129;

[0504] (21) Sequence numbers 130 to 134;

[0505] (22) Sequence numbers 135 to 139;

[0506] (23) Sequence numbers 140 to 144;

[0507] (24) Sequence numbers 145 to 149;

[0508] (25) Sequence numbers 150 to 154;

[0509] (26) Sequence numbers 155 to 159;

[0510] (27) Sequence numbers 160 to 164;

[0511] (28) Sequence numbers 165 to 169;

[0512] (29) Sequence numbers 170 to 174;

[0513] (30) Sequence numbers 175 to 179;

[0514] (31) Sequence numbers 180 to 184;

[0515] (32) Sequence numbers 185 to 189;

[0516] (33) Sequence numbers 190 to 194;

[0517] (34) Sequence numbers 195 to 199;

[0518] (35) Sequence numbers 200 to 204;

[0519] (36) Sequence numbers 205 to 210;

[0520] (37) Sequence numbers 211 to 216;

[0521] (38) Sequence numbers 217 to 222;

[0522] (39) Sequence numbers 223 to 228;

[0523] (40) Sequence numbers 229 to 234;

[0524] (41) Sequence numbers 235 to 240;

[0525] (42) Sequence numbers 241 to 246;

[0526] (43) Sequence numbers 247 to 252;

[0527] (44) Sequence numbers 253 to 258;

[0528] (45) Sequence numbers 259 to 264;

[0529] (46) Sequence numbers 265 to 270;

[0530] (47) Sequence numbers 271 to 276;

[0531] (48) Sequence numbers 277 to 282;

[0532] (49) Sequence numbers 283 to 288;

[0533] (50) Sequence numbers 289 to 294;

[0534] (51) Sequence numbers 295 to 300; and

[0535] (52) Sequence numbers 301 to 306.

[0536] The above zinc finger protein may comprise an amino acid sequence selected from the group consisting of SEQ ID NOs: 307 to 358.

[0537] The transcriptional regulatory sequence according to the present invention is highly effective in expressing the ZFP described above in Schwann cells from CMT patients. When expressed using the transcriptional regulatory sequence according to the present invention, the ZFP described above specifically inhibits the expression of the PMP22 protein induced by the P1 promoter compared to the P2 promoter. This is very useful for suppressing the expression of PMP22, which is pathologically relevant in CMT patients.

[0538] When the ZFP described above is expressed in Schwann cells of a CMT patient using the transcriptional regulatory sequence according to the present invention, the expression of the PMP22 protein by the P1 promoter may be reduced by 20% or more, 30% or more, or 40% or more compared to a normal person. For example, when the polynucleotide according to the present invention is expressed in Schwann cells of a CMT patient, the expression of the PMP22 protein by the P1 promoter may be reduced by 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or 80% compared to a normal person.

[0539] The transcription regulatory sequence according to the present invention can be used in a form that additionally includes a transcription repression domain and is operably linked to a target gene (e.g., a gene encoding a ZFP as described above).

[0540] A transcriptional repression domain refers to a protein sequence within a specific transcription factor or co-transcription factor that suppresses gene expression. This domain can act by interfering with the formation of the transcription initiation complex or by modulating histone modifications to condense chromatin. For example, the Krueppel-associated box (KRAB) domain is one such transcriptional repression domain, which recruits the histone deacetylase (HDAC) complex to suppress transcription.

[0541] Alternatively, the transcriptional regulatory sequence according to the present invention can be operably linked to a target gene (e.g., a gene encoding a ZFP as described above) without a transcriptional repression domain. The transcriptional repression domain may be, for example, a KRAB domain. The ZFP according to the present invention can effectively repress the expression of the PMP22 protein even without a transcriptional repression domain such as KRAB.

[0542] How to use

[0543] The PMP22 gene expression-regulating ZFP according to the present invention or a polynucleotide encoding the same can be used as a therapeutic agent for preventing or treating CMT disease. The ZFP according to the present invention can also be used in a form expressed by the transcriptional regulatory sequence described above.

[0544] Accordingly, the present invention provides a delivery vehicle useful as a gene therapy agent, comprising a polynucleotide encoding a ZFP as described herein. The delivery vehicle may comprise a transcriptional regulatory sequence as described herein suitable for expressing the ZFP in Schwann cells.

[0545] The vector that may contain the polynucleotide according to the present invention (a polynucleotide comprising a ZFP as described herein or comprising a ZFP and a transcriptional regulatory sequence as described herein) may be a plasmid vector or a viral vector. The viral vector may be, for example, a viral vector selected from the group consisting of a retrovirus, an adenovirus, a lentivirus, a poxvirus, a vaccinia virus, a herpesvirus, and an adeno-associated virus vector (AAV), but is not limited thereto. The viral vector may be, for example, a scAAV or a ssAAV, and preferably a scAAV.

[0546] The carrier that may contain the polynucleotide according to the present invention (a polynucleotide comprising a ZFP as described herein or comprising a ZFP and a transcriptional regulatory sequence as described herein) may be a carrier other than a plasmid or a virus, and may be selected from the group consisting of, but not limited to, liposomes, exosomes, lipid nanoparticles, polymer nanoparticles, extracellular vesicles, synthetic vesicles, and virus-like particles.

[0547] The polynucleotide included in the above delivery system (which comprises a ZFP as described herein, or a polynucleotide comprising a ZFP as described herein and a transcription regulatory sequence together) may or may not additionally include a transcription repression domain, for example, a KRAB domain. Since the ZFP according to the present invention can effectively repress the expression of the PMP22 protein even without a transcription repression domain such as KRAB, the overall size of the gene to be delivered can be reduced by not including a transcription repression domain such as KRAB, and also the possibility of affecting the expression of genes other than the target gene (off-target effect) or potential side effects can be minimized.

[0548] The present invention also provides a pharmaceutical composition for treating a disease associated with Schwann cells, comprising a ZFP as described herein or a polynucleotide encoding the same, and a delivery vehicle comprising the polynucleotide. The polynucleotide included in the pharmaceutical composition may comprise a transcriptional regulatory sequence as described herein. The polynucleotide may or may not comprise a transcriptional repression domain, such as KRAB.

[0549] The pharmaceutical composition described above can be used to treat diseases associated with Schwann cells. Because Schwann cells play a crucial role in the functional regulation, maintenance, and repair of the nervous system, normal Schwann cells are essential for proper nervous system function. Defects in Schwann cells are associated with a variety of diseases, including, but not limited to, multiple sclerosis, diabetic neuropathy, Guillain-Barré syndrome (GBS), Schwannoma, neurofibroma, malignant peripheral schwannoma, and chemotherapy (CMT). In the present invention, the disease associated with Schwann cells is preferably, but not necessarily limited to, CMT.

[0550] The pharmaceutical composition for treating a Schwann cell-related disease, preferably CMT, according to the present invention may, when administered to a patient suffering from a Schwann cell-related disease, preferably CMT, reduce the expression of PMP22 protein by the P1 promoter in the patient's Schwann cells by 20% or more, 30% or more, or 40% or more compared to a normal person. For example, the pharmaceutical composition for treating CMT according to the present invention may reduce the expression of PMP22 protein by the P1 promoter in the patient's Schwann cells by 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or 80% compared to a normal person.

[0551] The term "pharmaceutical composition" means a composition formulated for pharmaceutical use. In some embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier. Those skilled in the pharmaceutical art are familiar with pharmaceutical carriers that can be conventionally used to formulate a polynucleotide according to the invention (which comprises a ZFP as described herein, or which comprises a ZFP and a transcriptional regulatory sequence as described herein) for pharmaceutical use. In some embodiments, the pharmaceutical composition may comprise additional agents (e.g., agents for specific delivery, increased half-life, or other therapeutic compounds). The term "pharmaceutically acceptable carrier" means a pharmaceutically acceptable substance, composition, or vehicle, such as a liquid or solid filler, diluent, excipient, manufacturing aid, or solvent encapsulating material that carries or transports a compound from one site in the body (e.g., a delivery site) to another site (e.g., an organ, tissue, or part of the body). A pharmaceutically acceptable carrier is “acceptable” in the sense that it is compatible with the other ingredients of the formulation and not deleterious to the tissues of the subject (e.g., physiologically compatible, sterile, physiological pH, etc.). The terms “excipient,” “carrier,” “pharmaceutically acceptable carrier,” “vehicle,” and the like may be used interchangeably.

[0552] The pharmaceutical composition according to the present invention may also be referred to as a gene therapy agent. The gene therapy agent can deliver the polynucleotide according to the present invention (a polynucleotide comprising a ZFP as described herein, or a polynucleotide comprising a ZFP and a transcriptional regulatory sequence as described herein) to a patient in the form of a vector. Such vectors may be used by a method using a virus as a vector, a method using a virus-like vector, a non-viral method using a synthetic phospholipid or a synthetic cationic polymer, or an electroporation method that introduces a gene by applying an electric stimulus to a cell membrane. Among these, when a virus is used as a vector, for example, adeno-associated virus (AAV) can be used as a vector.

[0553] The above pharmaceutical composition or gene therapy agent may be administered via a route commonly used for administering gene therapy agents, such as parenteral administration.

[0554] The present invention also provides a method for treating a disease associated with Schwann cells, comprising administering a ZFP as described herein or a polynucleotide encoding the same, or a carrier comprising the polynucleotide. The polynucleotide used in the method of treatment may comprise a transcriptional regulatory sequence as described herein. The polynucleotide may or may not comprise a transcriptional repression domain such as KRAB. Diseases associated with Schwann cells include, but are not limited to, multiple sclerosis, diabetic neuropathy, Guillain-Barré syndrome (GBS), Schwannomas, neurofibromas, malignant peripheral neuropathy, and CMT. In the present invention, the disease associated with Schwann cells is preferably, but is not necessarily limited to, CMT.

[0555] The above treatment method may reduce the expression of the PMP22 protein driven by the P1 promoter in Schwann cells of a CMT patient by 20% or more, 30% or more, or 40% or more compared to a normal person. For example, the CMT treatment method according to the present invention may reduce the expression of the PMP22 protein driven by the P1 promoter in Schwann cells of a CMT patient by 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or 80% compared to a normal person.

[0556] The above treatment method involves administering to a subject in need thereof an effective amount of a polynucleotide according to the present invention (which comprises a ZFP as described herein, or a polynucleotide comprising a ZFP and a transcriptional regulatory sequence as described herein). As used herein, an "effective amount" refers to an amount of a biologically active agent sufficient to induce a desired biological response. In some embodiments, the effective amount is an amount necessary to ameliorate symptoms of the disease in an untreated patient. The effective amount of the active agent used in the therapeutic method and treatment of a disease may vary depending on the mode of administration, the age, weight, and general health of the subject. In one embodiment, the effective amount is an amount sufficient to significantly reduce the level of expression of PMP22 protein in a cell (e.g., in vitro, in vivo, or ex vivo). In one embodiment, the effective amount is an amount of the polynucleotide necessary to achieve a therapeutic effect (e.g., to reduce or control a symptom or condition of a CMT patient). In one embodiment, the effective amount is sufficient to ameliorate one or more symptoms of CMT.

[0557] The present invention may be as follows based on the above-described contents, but is not limited thereto.

[0558] 1. An isolated zinc finger protein comprising at least three zinc fingers and binding to the promoter or a part thereof of the PMP22 gene in a nervous system cell to reduce the expression of the PMP22 protein.

[0559] 2. An isolated zinc finger protein according to claim 1, wherein the promoter is a P1 promoter.

[0560] 3. An isolated zinc finger protein that specifically inhibits the expression of PMP 22 protein by the P1 promoter compared to the P2 promoter of the PMP22 gene in the first paragraph.

[0561] 4. An isolated zinc finger protein according to any one of claims 1 to 3, which binds to a nucleic acid sequence portion selected from the group consisting of SEQ ID NOs: 3 to 48 and the following sequences or a complementary sequence thereof.

[0562] 5'-CAGGGAGCA-3'

[0563] 5'-CACCAGGGA-3'

[0564] 5'-GGAGCACCA-3'

[0565] 5'-CAGGGAACA-3'

[0566] 5'-GGTGGTGCT-3'

[0567] 5'-CCTGTAACT-3'

[0568] 5'-CCTGTAACT-3'

[0569] 5'-AAGCCAGAC-3'

[0570] 5'-GTAACTGAA-3'

[0571] 5'-CCAGACCAG-3'

[0572] 5'-ACTGAAGCC-3'

[0573] 5'-GACCAGGCG-3'

[0574] 5. An isolated zinc finger protein comprising a set of amino acid sequences selected from the group consisting of (1) to (52) according to any one of claims 1 to 4:

[0575] (1) Sequence numbers 49 to 52;

[0576] (2) Sequence numbers 53 to 56;

[0577] (3) Sequence numbers 57 to 60;

[0578] (4) Sequence numbers 61 to 64;

[0579] (5) Sequence numbers 65 to 68;

[0580] (6) Sequence numbers 69 to 72;

[0581] (7) Sequence numbers 73 to 76;

[0582] (8) Sequence numbers 77 to 80;

[0583] (9) Sequence numbers 81 to 84;

[0584] (10) Sequence numbers 85 to 88;

[0585] (11) Sequence numbers 89 to 92;

[0586] (12) Sequence numbers 93 to 96;

[0587] (13) Sequence numbers 97 to 100;

[0588] (14) Sequence numbers 101 to 104;

[0589] (15) Sequence numbers 105 to 108;

[0590] (16) Sequence numbers 109 to 112;

[0591] (17) Sequence numbers 113 to 116;

[0592] (18) Sequence numbers 117 to 120;

[0593] (19) Sequence numbers 121 to 124;

[0594] (20) Sequence numbers 125 to 129;

[0595] (21) Sequence numbers 130 to 134;

[0596] (22) Sequence numbers 135 to 139;

[0597] (23) Sequence numbers 140 to 144;

[0598] (24) Sequence numbers 145 to 149;

[0599] (25) Sequence numbers 150 to 154;

[0600] (26) Sequence numbers 155 to 159;

[0601] (27) Sequence numbers 160 to 164;

[0602] (28) Sequence numbers 165 to 169;

[0603] (29) Sequence numbers 170 to 174;

[0604] (30) Sequence numbers 175 to 179;

[0605] (31) Sequence numbers 180 to 184;

[0606] (32) Sequence numbers 185 to 189;

[0607] (33) Sequence numbers 190 to 194;

[0608] (34) Sequence numbers 195 to 199;

[0609] (35) Sequence numbers 200 to 204;

[0610] (36) Sequence numbers 205 to 210;

[0611] (37) Sequence numbers 211 to 216;

[0612] (38) Sequence numbers 217 to 222;

[0613] (39) Sequence numbers 223 to 228;

[0614] (40) Sequence numbers 229 to 234;

[0615] (41) Sequence numbers 235 to 240;

[0616] (42) Sequence numbers 241 to 246;

[0617] (43) Sequence numbers 247 to 252;

[0618] (44) Sequence numbers 253 to 258;

[0619] (45) Sequence numbers 259 to 264;

[0620] (46) Sequence numbers 265 to 270;

[0621] (47) Sequence numbers 271 to 276;

[0622] (48) Sequence numbers 277 to 282;

[0623] (49) Sequence numbers 283 to 288;

[0624] (50) Sequence numbers 289 to 294;

[0625] (51) Sequence numbers 295 to 300; and

[0626] (52) Sequence numbers 301 to 306.

[0627] 6. An isolated zinc finger protein comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 307 to 358, according to any one of claims 1 to 5.

[0628] 7. An isolated zinc finger protein according to any one of claims 1 to 6, which reduces the expression of PMP22 protein by the P1 promoter in Schwann cells of Charcot-Marie-Tooth patients by 20% or more, 30% or more, or 40% or more compared to normal individuals.

[0629] 8. An isolated polynucleotide encoding an isolated zinc finger protein according to any one of claims 1 to 7.

[0630] 9. A polynucleotide according to claim 8, further comprising a Schwann cell-specific promoter operably linked to the polynucleotide.

[0631] 10. A polynucleotide according to claim 9, wherein the Schwann cell-specific promoter is an MPZ promoter, an MBP promoter, a PMP22 promoter, or a MAG promoter.

[0632] 11. A polynucleotide according to any one of claims 8 to 10, further comprising a transcription repression domain.

[0633] 12. A polynucleotide according to claim 11, wherein the transcription repression domain is a KRAB (Krueppel-associated box) domain.

[0634] 13. A polynucleotide according to any one of claims 8 to 10, which does not additionally comprise a transcription repression domain.

[0635] 14. A polynucleotide according to claim 13, wherein the transcription repression domain is a KRAB domain.

[0636] 15. A polynucleotide comprising a transcriptional regulatory sequence comprising a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or at least 100% sequence identity with a nucleotide sequence selected from the group consisting of SEQ ID NOs: 363 to 366.

[0637] 16. A polynucleotide comprising a transcriptional regulatory sequence comprising a nucleotide sequence having a sequence identity of at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or at least 100% with the nucleotide sequence of SEQ ID NO: 365 or 366 in accordance with claim 15.

[0638] 17. A polynucleotide according to claim 15 or 16, wherein the transcription regulatory sequence further comprises one or more enhancer sequences.

[0639] 18. A polynucleotide according to claim 17, wherein at least one enhancer has a sequence identity of at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or at least 100% with a nucleotide sequence independently selected from the group consisting of SEQ ID NOs: 359 to 362.

[0640] 19. A polynucleotide according to claim 15, wherein the transcription regulatory sequence comprises a nucleotide sequence having a sequence identity of at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or at least 100% with a nucleotide sequence selected from the group consisting of SEQ ID NOs: 367 to 374.

[0641] 20. A polynucleotide according to any one of claims 15 to 19, further comprising a target gene sequence, wherein the target gene is a nucleic acid encoding a gene to be expressed in a Schwann cell or a protein to be expressed in a Schwann cell, and is operably linked to a transcriptional regulatory sequence.

[0642] 21. A polynucleotide according to claim 20, wherein the target gene is a nucleic acid encoding a zinc finger protein that binds to the promoter of the PMP22 gene or a part thereof in a nervous system cell and reduces the expression of the PMP22 protein.

[0643] 22. A polynucleotide according to claim 21, wherein the zinc finger protein specifically inhibits the expression of the PMP 22 protein by the P1 promoter compared to the P2 promoter of the PMP22 gene.

[0644] 23. A polynucleotide according to claim 21 or 22, further comprising a transcription repression domain.

[0645] 24. A polynucleotide according to claim 23, wherein the transcription repression domain is a KRAB (Krueppel-associated box) domain.

[0646] 25. A polynucleotide according to claim 21 or 22, which does not additionally comprise a transcription repression domain.

[0647] 26. A polynucleotide according to claim 25, wherein the transcription repression domain is a KRAB domain.

[0648] 27. A polynucleotide according to any one of claims 21 to 26, wherein the zinc finger protein binds to a nucleic acid sequence portion selected from the group consisting of SEQ ID NOs: 3 to 48 and the following sequences or a complementary sequence thereof.

[0649] 5'-CAGGGAGCA-3'

[0650] 5'-CACCAGGGA-3'

[0651] 5'-GGAGCACCA-3'

[0652] 5'-CAGGGAACA-3'

[0653] 5'-GGTGGTGCT-3'

[0654] 5'-CCTGTAACT-3'

[0655] 5'-CCTGTAACT-3'

[0656] 5'-AAGCCAGAC-3'

[0657] 5'-GTAACTGAA-3'

[0658] 5'-CCAGACCAG-3'

[0659] 5'-ACTGAAGCC-3'

[0660] 5'-GACCAGGCG-3'

[0661] 28. A polynucleotide according to any one of claims 21 to 27, wherein the zinc finger protein comprises a set of amino acid sequences selected from the group consisting of (1) to (52):

[0662] (1) Sequence numbers 49 to 52;

[0663] (2) Sequence numbers 53 to 56;

[0664] (3) Sequence numbers 57 to 60;

[0665] (4) Sequence numbers 61 to 64;

[0666] (5) Sequence numbers 65 to 68;

[0667] (6) Sequence numbers 69 to 72;

[0668] (7) Sequence numbers 73 to 76;

[0669] (8) Sequence numbers 77 to 80;

[0670] (9) Sequence numbers 81 to 84;

[0671] (10) Sequence numbers 85 to 88;

[0672] (11) Sequence numbers 89 to 92;

[0673] (12) Sequence numbers 93 to 96;

[0674] (13) Sequence numbers 97 to 100;

[0675] (14) Sequence numbers 101 to 104;

[0676] (15) Sequence numbers 105 to 108;

[0677] (16) Sequence numbers 109 to 112;

[0678] (17) Sequence numbers 113 to 116;

[0679] (18) Sequence numbers 117 to 120;

[0680] (19) Sequence numbers 121 to 124;

[0681] (20) Sequence numbers 125 to 129;

[0682] (21) Sequence numbers 130 to 134;

[0683] (22) Sequence numbers 135 to 139;

[0684] (23) Sequence numbers 140 to 144;

[0685] (24) Sequence numbers 145 to 149;

[0686] (25) Sequence numbers 150 to 154;

[0687] (26) Sequence numbers 155 to 159;

[0688] (27) Sequence numbers 160 to 164;

[0689] (28) Sequence numbers 165 to 169;

[0690] (29) Sequence numbers 170 to 174;

[0691] (30) Sequence numbers 175 to 179;

[0692] (31) Sequence numbers 180 to 184;

[0693] (32) Sequence numbers 185 to 189;

[0694] (33) Sequence numbers 190 to 194;

[0695] (34) Sequence numbers 195 to 199;

[0696] (35) Sequence numbers 200 to 204;

[0697] (36) Sequence numbers 205 to 210;

[0698] (37) Sequence numbers 211 to 216;

[0699] (38) Sequence numbers 217 to 222;

[0700] (39) Sequence numbers 223 to 228;

[0701] (40) Sequence numbers 229 to 234;

[0702] (41) Sequence numbers 235 to 240;

[0703] (42) Sequence numbers 241 to 246;

[0704] (43) Sequence numbers 247 to 252;

[0705] (44) Sequence numbers 253 to 258;

[0706] (45) Sequence numbers 259 to 264;

[0707] (46) Sequence numbers 265 to 270;

[0708] (47) Sequence numbers 271 to 276;

[0709] (48) Sequence numbers 277 to 282;

[0710] (49) Sequence numbers 283 to 288;

[0711] (50) Sequence numbers 289 to 294;

[0712] (51) Sequence numbers 295 to 300; and

[0713] (52) Sequence numbers 301 to 306.

[0714] 29. A polynucleotide according to claim 28, wherein the zinc finger protein comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 307 to 358.

[0715] 30. A polynucleotide according to any one of claims 21 to 29, which reduces the expression of PMP22 protein by 20% or more, 30% or more, or 40% or more when expressed in Schwann cells of a Charcot-Marie-Tooth patient.

[0716] 31. A delivery vehicle comprising a polynucleotide according to any one of claims 8 to 30.

[0717] 32. A vector according to claim 31, which is a plasmid vector, a viral vector or a non-viral vector.

[0718] 33. A vector according to claim 32, which is a viral vector selected from the group consisting of retrovirus, adenovirus, lentivirus, poxvirus, vaccinia virus, herpesvirus, and adeno-associated virus vector (AAV).

[0719] 34. A delivery vehicle in claim 32, which is a non-viral vector selected from the group consisting of liposomes, exosomes, lipid nanoparticles, polymer nanoparticles, extracellular vesicles, synthetic vesicles, and virus-like particles.

[0720] 35. A pharmaceutical composition for treating a disease associated with Schwann cells, comprising a zinc finger protein according to any one of claims 1 to 7, a polynucleotide according to any one of claims 8 to 30, or a transporter according to any one of claims 31 to 34.

[0721] 36. A pharmaceutical composition according to claim 35, wherein the disease associated with Schwann cells is Charcot-Marie-Tooth disease.

[0722] 37. A pharmaceutical composition according to claim 36, which, when administered to a Charcot-Marie-Tooth patient, reduces the expression of PMP22 protein by the P1 promoter in Schwann cells by 20% or more, 30% or more, or 40% or more compared to a normal person.

[0723] 38. A method for treating a disease associated with Schwann cells, comprising administering to a patient in need of treatment for Charcot-Marie-Tooth disease a zinc finger protein according to any one of claims 1 to 7, a polynucleotide according to any one of claims 8 to 30, or a carrier according to any one of claims 31 to 34.

[0724] 39. A method of treating, in claim 38, a disease associated with Schwann cells being Charcot-Marie-Tooth disease.

[0725] 40. A method of treating, in claim 39, wherein the expression of PMP22 protein by the P1 promoter in Schwann cells of a Charcot-Marie-Tooth patient is reduced by 20% or more, 30% or more, or 40% or more compared to a normal person.

[0726] Hereinafter, the present invention will be described in detail with reference to the following examples. However, the following examples are provided only to illustrate the present invention and the present invention is not limited thereto.

[0727]

[0728] Example 1: Production of ZFP construct for regulating PMP22 gene transcription

[0729] By identifying a ZFP-binding DNA sequence in the P1 promoter of the PMP22 gene, ZFPs consisting of 4- to 6-finger sequences were designed using zinc finger motifs that recognize 3 bp. In addition, 3-finger ZFPs capable of skipping 1 bp and base-skipping ZFPs in the form of linked 3-finger ZFPs (termed "sZFPs") were designed. To construct a vector expressing the designed ZFPs, ZFPs expressed under the CMV promoter were constructed. Each zinc finger motif was connected with a "TEGKP" linker, and the 1-bp skipping site was connected using a "LRQKDGERP" linker. The C-terminus of ZFP was fused to the c-myc NLS (nuclear export signal) and FLAG tag, so that the expressed ZFPs can enter the nucleus and the expression of ZFPs can be confirmed through Western blotting. The manufactured ZFPs were linked in the following order: [wild type N-terminal domain]-[ZFP]-[NLS (c-Myc)]-[tag (1x FLAG)]. The coding sequences of the proteins used are located between the CMV promoter and T7 promoter and the terminator sequence.

[0730] The amino acid sequences of the manufactured ZFPs are as follows.

[0731] ZFP1

[0732] MRKYPNRPSKTPPHERPFECKDCGKAFIQKSNLIRHQRTHTGEKPYKCPECGKSFSTKNSLTEHQRTHTGEKPYTCSDCGKAFRDKSCLNRHRRTHTGEKPYKCPECGKSFSTSHSLTEHQRTHTGEKPAAKRVKLDDYKDDDDK

[0733] ZFP2

[0734] MRKYPNRPSKTPPHERPYECVQCGKGFTQSSNLITHQRVHTGEKPFQCKTCQRKFSRSDHLKTHTRTHTGEKPFQCRICMRNFSDSGNLRVHIRTHTGEKPYTCSYCGKSFTQSNTLKQHTRIHTGEKPAAKRVKLDDYKDDDDK

[0735] ZFP3

[0736] MRKYPNRPSKTPPHERPYTCSDCGKAFRDKSCLNRHRRTHTGEKPYRCKYCDRSFSDSSNLQRHVRNIHTGEKPYECVQCGKGFTQSSNLITHQRVHTGEKPFQCKTCQRKFSRSDHLKTHTRTHTGEKPAAKRVKLDDYKDDDDK

[0737] ZFP4

[0738] MRKYPNRPSKTPPHERPFQCKTCQRKFSRSDHLKTHTRTHTGEKPYTCSDCGKAFRDKSCLNRHRRTHTGEKPYRCKYCDRSFSDSSNLQRHVRNIHTGEKPYECVQCGKGFTQSSNLITHQRVHTGEKPAAKRVKLDDYKDDDDK

[0739] ZFP5

[0740] MRKYPNRPSKTPPHERPYKCPECGKSFSTSHSLTEHQRTHTGEKPYKCPDCGKSFSQSSSLIRHQRTHTGEKPYKCGQCGKFYSQVSHLTRHQKIHTGEKPYKCPECGKSFSRADNLTEHQRTHTGEKPAAKRVKLDDYKDDDDK

[0741] ZFP6

[0742] MRKYPNRPSKTPPHERPYKCPECGKSFSSKKALTEHQRTHTGEKPYKCPECGKSFSSKKALTEHQRTHTGEKPYGCHLCGKAFSKSSNLRRHEMIHTGEKPFQCKTCQRKFSRSDHLKTHTRTHTGEKPAAKRVKLDDYKDDDDK

[0743] ZFP7

[0744] MRKYPNRPSKTPPHERPYKCMECGKAFNRRSHLTRHQRIHTGEKPYKCPECGKSFSTSHSLTEHQRTHTGEKPYKCPECGKSFSTSHSLTEHQRTHTGEKPYKCPDCGKSFSQSSSLIRHQRTHTGEKPAAKRVKLDDYKDDDDK

[0745] ZFP8

[0746] MRKYPNRPSKTPPHERPYKCGQCGKFYSQVSHLTRHQKIHTGEKPYKCPECGKSFSRADNLTEHQRTHTGEKPYKCPECGKSFSSKKALTEHQRTHTGEKPYKCPECGKSFSSKKALTEHQRTHTGEKPAAKRVKLDDYKDDDDK

[0747] ZFP9

[0748] MRKYPNRPSKTPPHERPFQCRICMRNFSDSGNLRVHIRTHTGEKPYKCMECGKAFNRRSHLTRHQRIHTGEKPYKCPECGKSFSTSHSLTEHQRTHTGEKPYKCPECGKSFSTSHSLTEHQRTHTGEKPAAKRVKLDDYKDDDDK

[0749] ZFP10

[0750] MRKYPNRPSKTPPHERPYKCPECGKSFSSPADLTRHQRTHTGEKPYKCGQCGKFYSQVSHLTRHQKIHTGEKPYKCPECGKSFSRADNLTEHQRTHTGEKPYKCPECGKSFSSKKALTEHQRTHTGEKPAAKRVKLDDYKDDDDK

[0751] ZFP11

[0752] MRKYPNRPSKTPPHERPYECNYCGKTFSVSSTLIRHQRIHTGEKPYRCEECGKAFRWPSNLTRHKRIHTGEKPYRCEECGKAFRWPSNLTRHKRIHTGEKPYKCPECGKSFSTKNSLTEHQRTHTGEKPAAKRVKLDDYKDDDDK

[0753] ZFP12

[0754] MRKYPNRPSKTPPHERPFECKDCGKAFIQKSNLIRHQRTHTGEKPYKCPECGKSFSTHLDLIRHQRTHTGEKPYKCPDCGKSFSQSSSLIRHQRTHTGEKPYKCPECGKSFSTKNSLTEHQRTHTGEKPAAKRVKLDDYKDDDDK

[0755] ZFP13

[0756] MRKYPNRPSKTPPHERPYTCSDCGKAFRDKSCLNRHRRTHTGEKPFECKDCGKAFIQKSNLIRHQRTHTGEKPYKCPECGKSFSTHLDLIRHQRTHTGEKPYKCPDCGKSFSQSSSLIRHQRTHTGEKPAAKRVKLDDYKDDDDK

[0757] ZFP14

[0758] MRKYPNRPSKTPPHERPYECHDCGKSFRQSTHLTQHRRIHTGEKPYTCSDCGKAFRDKSCLNRHRRTHTGEKPFECKDCGKAFIQKSNLIRHQRTHTGEKPYKCPECGKSFSTHLDLIRHQRTHTGEKPAAKRVKLDDYKDDDDK

[0759] ZFP15

[0760] MRKYPNRPSKTPPHERPYKCPECGKSFSTSHSLTEHQRTHTGEKPYECHDCGKSFRQSTHLTQHRRIHTGEKPYTCSDCGKAFRDKSCLNRHRRTHTGEKPFECKDCGKAFIQKSNLIRHQRTHTGEKPAAKRVKLDDYKDDDDK

[0761] ZFP16

[0762] MRKYPNRPSKTPPHERPYKCPECGKSFSRADNLTEHQRTHTGEKPYRCKYCDRSFSDSSNLQRHVRNIHTGEKPYKCPECGKSFSTSHSLTEHQRTHTGEKPFACPECPKRFMRSDNLTQHIKTHTGEKPAAKRVKLDDYKDDDDK

[0763] ZFP17

[0764] MRKYPNRPSKTPPHERPYKCPECGKSFSDPGHLVRHQRTHTGEKPYKCPECGKSFSTSHSLTEHQRTHTGEKPYECHDCGKSFRQSTHLTQHRRIHTGEKPYTCSDCGKAFRDKSCLNRHRRTHTGEKPAAKRVKLDDYKDDDDK

[0765] ZFP18

[0766] MRKYPNRPSKTPPHERPYHCDWDGCGWKFARSDELTRHYRKHTGEKPYKCPECGKSFSRADNLTEHQRTHTGEKPYRCKYCDRSFSDSSNLQRHVRNIHTGEKPYKCPECGKSFSTSHSLTEHQRTHTGEKPAAKRVKLDDYKDDDDK

[0767] ZFP19

[0768] MRKYPNRPSKTPPHERPYSCGICGKSFSDSSAKRRHCILHTGEKPYKCPECGKSFSDPGHLVRHQRTHTGEKPYKCPECGKSFSTSHSLTEHQRTHTGEKPYECHDCGKSFRQSTHLTQHRRIHTGEKPAAKRVKLDDYKDDDDK

[0769] ZFP20

[0770] MRKYPNRPSKTPPHERPYECVQCGKGFTQSSNLITHQRVHTGEKPFQCKTCQRKFSRSDHLKTHTRTHTGEKPFQCRICMRNFSDSGNLRVHIRTHTGEKPYTCSYCGKSFTQSNTLKQHTRIHTGEKPYECDHCGKSFSQSSHLNVHKRTHTGEKPAAKRVKLDDYKDDDDK

[0771] ZFP21

[0772] MRKYPNRPSKTPPHERPYRCKYCDRSFSDSSNLQRHVRNIHTGEKPYECVQCGKGFTQSSNLITHQRVHTGEKPFQCKTCQRKFSRSDHLKTHTRTHTGEKPFQCRICMRNFSDSGNLRVHIRTHTGEKPYTCSYCGKSFTQSNTLKQHTRIHTGEKPAAKRVKLDDYKDDDDK

[0773] ZFP22

[0774] MRKYPNRPSKTPPHERPYTCSDCGKAFRDKSCLNRHRRTHTGEKPYRCKYCDRSFSDSSNLQRHVRNIHTGEKPYECVQCGKGFTQSSNLITHQRVHTGEKPFQCKTCQRKFSRSDHLKTHTRTHTGEKPFQCRICMRNFSDSGNLRVHIRTHTGEKPAAKRVKLDDYKDDDDK

[0775] ZFP23

[0776] MRKYPNRPSKTPPHERPFQCKTCQRKFSRSDHLKTHTRTHTGEKPYTCSDCGKAFRDKSCLNRHRRTHTGEKPYRCKYCDRSFSDSSNLQRHVRNIHTGEKPYECVQCGKGFTQSSNLITHQRVHTGEKPFQCKTCQRKFSRSDHLKTHTRTHTGEKPAAKRVKLDDYKDDDDK

[0777] ZFP24

[0778] MRKYPNRPSKTPPHERPYKCPECGKSFSTSHSLTEHQRTHTGEKPYKCPECGKSFSTSHSLTEHQRTHTGEKPYKCPDCGKSFSQSSSLIRHQRTHTGEKPYKCGQCGKFYSQVSHLTRHQKIHTGEKPYKCPECGKSFSRADNLTEHQRTHTGEKPAAKRVKLDDYKDDDDK

[0779] ZFP25

[0780] MRKYPNRPSKTPPHERPYKCPECGKSFSRADNLTEHQRTHTGEKPYKCPECGKSFSSKKALTEHQRTHTGEKPYKCPECGKSFSSKKALTEHQRTHTGEKPYGCHLCGKAFSKSSNLRRHEMIHTGEKPFQCKTCQRKFSRSDHLKTHTRTHTGEKPAAKRVKLDDYKDDDDK

[0781] ZFP26

[0782] MRKYPNRPSKTPPHERPYKCMECGKAFNRRSHLTRHQRIHTGEKPYKCPECGKSFSTSHSLTEHQRTHTGEKPYKCPECGKSFSTSHSLTEHQRTHTGEKPYKCPDCGKSFSQSSSLIRHQRTHTGEKPYKCGQCGKFYSQVSHLTRHQKIHTGEKPAAKRVKLDDYKDDDDK

[0783] ZFP27

[0784] MRKYPNRPSKTPPHERPYKCGQCGKFYSQVSHLTRHQKIHTGEKPYKCPECGKSFSRADNLTEHQRTHTGEKPYKCPECGKSFSSKKALTEHQRTHTGEKPYKCPECGKSFSSKKALTEHQRTHTGEKPYGCHLCGKAFSKSSNLRRHEMIHTGEKPAAKRVKLDDYKDDDDK

[0785] ZFP28

[0786] MRKYPNRPSKTPPHERPFQCRICMRNFSDSGNLRVHIRTHTGEKPYKCMECGKAFNRRSHLTRHQRIHTGEKPYKCPECGKSFSTSHSLTEHQRTHTGEKPYKCPECGKSFSTSHSLTEHQRTHTGEKPYKCPDCGKSFSQSSSLIRHQRTHTGEKPAAKRVKLDDYKDDDDK

[0787] ZFP29

[0788] MRKYPNRPSKTPPHERPYKCPECGKSFSSPADLTRHQRTHTGEKPYKCGQCGKFYSQVSHLTRHQKIHTGEKPYKCPECGKSFSRADNLTEHQRTHTGEKPYKCPECGKSFSSKKALTEHQRTHTGEKPYKCPECGKSFSSKKALTEHQRTHTGEKPAAKRVKLDDYKDDDDK

[0789] ZFP30

[0790] MRKYPNRPSKTPPHERPYTCSDCGKAFRDKSCLNRHRRTHTGEKPFECKDCGKAFIQKSNLIRHQRTHTGEKPYKCPECGKSFSTHLDLIRHQRTHTGEKPYKCPDCGKSFSQSSSLIRHQRTHTGEKPYKCPECGKSFSTKNSLTEHQRTHTGEKPAAKRVKLDDYKDDDDK

[0791] ZFP31

[0792] MRKYPNRPSKTPPHERPYECHDCGKSFRQSTHLTQHRRIHTGEKPYTCSDCGKAFRDKSCLNRHRRTHTGEKPFECKDCGKAFIQKSNLIRHQRTHTGEKPYKCPECGKSFSTHLDLIRHQRTHTGEKPYKCPDCGKSFSQSSSLIRHQRTHTGEKPAAKRVKLDDYKDDDDK

[0793] ZFP32

[0794] MRKYPNRPSKTPPHERPYKCPECGKSFSTSHSLTEHQRTHTGEKPYECHDCGKSFRQSTHLTQHRRIHTGEKPYTCSDCGKAFRDKSCLNRHRRTHTGEKPFECKDCGKAFIQKSNLIRHQRTHTGEKPYKCPECGKSFSTHLDLIRHQRTHTGEKPAAKRVKLDDYKDDDDK

[0795] ZFP33

[0796] MRKYPNRPSKTPPHERPYKCPECGKSFSDPGHLVRHQRTHTGEKPYKCPECGKSFSTSHSLTEHQRTHTGEKPYECHDCGKSFRQSTHLTQHRRIHTGEKPYTCSDCGKAFRDKSCLNRHRRTHTGEKPFECKDCGKAFIQKSNLIRHQRTHTGEKPAAKRVKLDDYKDDDDK

[0797] ZFP34

[0798] MRKYPNRPSKTPPHERPYHCDWDGCGWKFARSDELTRHYRKHTGEKPYKCPECGKSFSRADNLTEHQRTHTGEKPYRCKYCDRSFSDSSNLQRHVRNIHTGEKPYKCPECGKSFSTSHSLTEHQRTHTGEKPFACPECPKRFMRSDNLTQHIKTHTGEKPAAKRVKLDDYKDDDDK

[0799] ZFP35

[0800] MRKYPNRPSKTPPHERPYSCGICGKSFSDSSAKRRHCILHTGEKPYKCPECGKSFSDPGHLVRHQRTHTGEKPYKCPECGKSFSTSHSLTEHQRTHTGEKPYECHDCGKSFRQSTHLTQHRRIHTGEKPYTCSDCGKAFRDKSCLNRHRRTHTGEKPAAKRVKLDDYKDDDDK

[0801] ZFP36

[0802] MRKYPNRPSKTPPHERPYRCKYCDRSFSDSSNLQRHVRNIHTGEKPYECVQCGKGFTQSSNLITHQRVHTGEKPFQCKTCQRKFSRSDHLKTHTRTHTGEKPFQCRICMRNFSDSGNLRVHIRTHTGEKPYTCSYCGKSFTQSNTLKQHTRIHTGEKPYECDHCGKSFSQSSHLNVHKRTHTGEKPAAKRVKLDDYKDDDDK

[0803] ZFP37

[0804] MRKYPNRPSKTPPHERPYTCSDCGKAFRDKSCLNRHRRTHTGEKPYRCKYCDRSFSDSSNLQRHVRNIHTGEKPYECVQCGKGFTQSSNLITHQRVHTGEKPFQCKTCQRKFSRSDHLKTHTRTHTGEKPFQCRICMRNFSDSGNLRVHIRTHTGEKPYTCSYCGKSFTQSNTLKQHTRIHTGEKPAAKRVKLDDYKDDDDK

[0805] ZFP38

[0806] MRKYPNRPSKTPPHERPFQCKTCQRKFSRSDHLKTHTRTHTGEKPYTCSDCGKAFRDKSCLNRHRRTHTGEKPYRCKYCDRSFSDSSNLQRHVRNIHTGEKPYECVQCGKGFTQSSNLITHQRVHTGEKPFQCKTCQRKFSRSDHLKTHTRTHTGEKPFQCRICMRNFSDSGNLRVHIRTHTGEKPAAKRVKLDDYKDDDDK

[0807] ZFP39

[0808] MRKYPNRPSKTPPHERPYKCMECGKAFNRRSHLTRHQRIHTGEKPYKCPECGKSFSTSHSLTEHQRTHTGEKPYKCPECGKSFSTSHSLTEHQRTHTGEKPYKCPDCGKSFSQSSSLIRHQRTHTGEKPYKCGQCGKFYSQVSHLTRHQKIHTGEKPYKCPECGKSFSRADNLTEHQRTHTGEKPAAKRVKLDDYKDDDDK

[0809] ZFP40

[0810] MRKYPNRPSKTPPHERPYKCGQCGKFYSQVSHLTRHQKIHTGEKPYKCPECGKSFSRADNLTEHQRTHTGEKPYKCPECGKSFSSKKALTEHQRTHTGEKPYKCPECGKSFSSKKALTEHQRTHTGEKPYGCHLCGKAFSKSSNLRRHEMIHTGEKPFQCKTCQRKFSRSDHLKTHTRTHTGEKPAAKRVKLDDYKDDDDK

[0811] ZFP41

[0812] MRKYPNRPSKTPPHERPFQCRICMRNFSDSGNLRVHIRTHTGEKPYKCMECGKAFNRRSHLTRHQRIHTGEKPYKCPECGKSFSTSHSLTEHQRTHTGEKPYKCPECGKSFSTSHSLTEHQRTHTGEKPYKCPDCGKSFSQSSSLIRHQRTHTGEKPYKCGQCGKFYSQVSHLTRHQKIHTGEKPAAKRVKLDDYKDDDDK

[0813] ZFP42

[0814] MRKYPNRPSKTPPHERPYKCPECGKSFSSPADLTRHQRTHTGEKPYKCGQCGKFYSQVSHLTRHQKIHTGEKPYKCPECGKSFSRADNLTEHQRTHTGEKPYKCPECGKSFSSKKALTEHQRTHTGEKPYKCPECGKSFSSKKALTEHQRTHTGEKPYGCHLCGKAFSKSSNLRRHEMIHTGEKPAAKRVKLDDYKDDDDK

[0815] ZFP43

[0816] MRKYPNRPSKTPPHERPYECHDCGKSFRQSTHLTQHRRIHTGEKPYTCSDCGKAFRDKSCLNRHRRTHTGEKPFECKDCGKAFIQKSNLIRHQRTHTGEKPYKCPECGKSFSTHLDLIRHQRTHTGEKPYKCPDCGKSFSQSSSLIRHQRTHTGEKPYKCPECGKSFSTKNSLTEHQRTHTGEKPAAKRVKLDDYKDDDDK

[0817] ZFP44

[0818] MRKYPNRPSKTPPHERPYKCPECGKSFSTSHSLTEHQRTHTGEKPYECHDCGKSFRQSTHLTQHRRIHTGEKPYTCSDCGKAFRDKSCLNRHRRTHTGEKPFECKDCGKAFIQKSNLIRHQRTHTGEKPYKCPECGKSFSTHLDLIRHQRTHTGEKPYKCPDCGKSFSQSSSLIRHQRTHTGEKPAAKRVKLDDYKDDDDK

[0819] ZFP45

[0820] MRKYPNRPSKTPPHERPYKCPECGKSFSDPGHLVRHQRTHTGEKPYKCPECGKSFSTSHSLTEHQRTHTGEKPYECHDCGKSFRQSTHLTQHRRIHTGEKPYTCSDCGKAFRDKSCLNRHRRTHTGEKPFECKDCGKAFIQKSNLIRHQRTHTGEKPYKCPECGKSFSTHLDLIRHQRTHTGEKPAAKRVKLDDYKDDDDK

[0821] ZFP46

[0822] MRKYPNRPSKTPPHERPYSCGICGKSFSDSSAKRRHCILHTGEKPYKCPECGKSFSDPGHLVRHQRTHTGEKPYKCPECGKSFSTSHSLTEHQRTHTGEKPYECHDCGKSFRQSTHLTQHRRIHTGEKPYTCSDCGKAFRDKSCLNRHRRTHTGEKPFECKDCGKAFIQKSNLIRHQRTHTGEKPAAKRVKLDDYKDDDDK

[0823] ZFPs1 (=sZFP1)

[0824] MRKYPNRPSKTPPHERPYKCGQCGKFYSQVSHLTRHQKIHTGEKPYKCPECGKSFSRADNLTEHQRTHTGEKPYKCPECGKSFSSKKALTEHQRTHLRQKDGERPYKCPDCGKSFSQSSSLIRHQRTHTGEKPYKCGQCGKFYSQVSHLTRHQKIHTGEKPYKCPECGKSFSRADNLTEHQRTHTGEKPAAKRVKLDDYKDDDDK

[0825] ZFPs3 (=sZFP3)

[0826] MRKYPNRPSKTPPHERPYKCPECGKSFSSPADLTRHQRTHTGEKPYKCGQCGKFYSQVSHLTRHQKIHTGEKPYKCPECGKSFSRADNLTEHQRTHLRQKDGERPYKCPECGKSFSTSHSLTEHQRTHTGEKPYKCPDCGKSFSQSSSLIRHQRTHTGEKPYKCGQCGKFYSQVSHLTRHQKIHTGEKPAAKRVKLDDYKDDDDK

[0827] ZFPs5 (=sZFP5)

[0828] MRKYPNRPSKTPPHERPYKCPECGKSFSTHLDLIRHQRTHTGEKPYKCPDCGKSFSQSSSLIRHQRTHTGEKPYKCPECGKSFSTKNSLTEHQRTHLRQKDGERPYECNYCGKTFSVSSTLIRHQRIHTGEKPYRCEECGKAFRWPSNLTRHKRIHTGEKPYRCEECGKAFRWPSNLTRHKRIHTGEKPAAKRVKLDDYKDDDDK

[0829] ZFPs7 (=sZFP7)

[0830] MRKYPNRPSKTPPHERPYRCKYCDRSFSDSSNLQRHVRNIHTGEKPYKCPECGKSFSTSHSLTEHQRTHTGEKPFACPECPKRFMRSDNLTQHIKTHLRQKDG ERPYKCPECGKSFSTHLDLIRHQRTHTGEKPYKCPDCGKSFSQSSSLIRHQRTHTGEKPYKCPECGKSFSTKNSLTEHQRTHTGEKPAAKRVKLDDYKDDDDK

[0831] ZFPs9 (=sZFP9)

[0832] MRKYPNRPSKTPPHERPYKCPECGKSFSRADNLTEHQRTHTGEKPYRCKYCDRSFSDSSNLQRHVRNIHTGEKPYKCPECGKSFSTSHSLTEHQRTHLRQKDG ERPFECKDCGKAFIQKSNLIRHQRTHTGEKPYKCPECGKSFSTHLDLIRHQRTHTGEKPYKCPDCGKSFSQSSSLIRHQRTHTGEKPAAKRVKLDDYKDDDDK

[0833] ZFPs12 (=sZFP12)

[0834] MRKYPNRPSKTPPHERPYHCDWDGCGWKFARSDELTRHYRKHTGEKPYKCPECGKSFSRADNLTEHQRTHTGEKPYRCKYCDRSFSDSSNLQRHVRNIHLRQKD GERPYTCSDCGKAFRDKSCLNRHRRTHTGEKPFECKDCGKAFIQKSNLIRHQRTHTGEKPYKCPECGKSFSTHLDLIRHQRTHTGEKPAAKRVKLDDYKDDDDK

[0835] The nucleic acid sequences to which each of these ZFPs bind are shown in Figure 1.

[0836] Example 2: Production of a PMP22 gene P1 promoter activity reporter system.

[0837] To confirm whether the manufactured ZFPs bind to the P1 promoter sequence of the PMP22 gene, genomic DNA of a human lymphoblastoid cell line was extracted, and a 1.1 kb region of the P1 promoter of the PMP22 gene was subjected to nested PCR. An expression vector was constructed by cloning the PMP22-p1 promoter sequence into the pGL3.0-luciferase (firefly) vector.

[0838] Example 3: Measurement of transcriptional regulation efficiency of the PMP22 gene by ZFP using an in vitro reporter system.

[0839] HEK293T cell lines and sNF02.2 cell lines were seeded at 0.75 x 10 per well in a 48-well plate. 5 Dog and 0.18 x 10 5 The cells were divided into 1 μg (0.5 μg ZFP + 0.5 μg reporter) and incubated at room temperature for 15 minutes by mixing 1.5 μL Lipofectamine 2000 and 1 μL TransfeX with a total of 1 μg of plasmid (0.5 μg ZFP + 0.5 μg reporter). The mixture was then incubated at room temperature for 15 minutes. 48 hours after plasmid introduction, the transcriptional regulation efficiency of the PMP22 gene by ZFP was measured using a luciferase activity assay kit and a luminometer.

[0840] The results are shown in Table 2 below and also in Figure 2.

[0841] Transcription efficiency compared to ZFP ID control (%) Transcription efficiency compared to ZFP ID control (%) HEK293TsNF02.2 HEK293TsNF02.2 ZFP1111.4861.46 ZFP2739.499.81 ZFP268.0162.42 ZFP2877.1827.53 ZFP343.1333.35 ZFP2923.215.10 ZFP424.7715.71 ZFP3046.0116.38 ZFP518.8814.43 ZFP3123.2110.11 ZFP639.3131.43 ZFP3233.3730 .22ZFP786.7263.89ZFP3319.3021.06ZFP845.8242.80ZFP3449.5834.79ZFP958.4090.13ZFP3564.1045.08ZFP1033.60 29.38ZFP3687.7527.47ZFP1153.5370.79ZFP3758.8810.88ZFP1272.0487.84ZFP3832.8213.79ZFP1358.1728.69ZFP392 0.548.66ZFP1470.3854.45ZFP4037.3328.58ZFP1546.5734.20ZFP4160.2231.50ZFP16112.2954.56ZFP4223.5214.02Z FP1791.2242.44ZFP4325.5722.53ZFP1894.8567.70ZFP4421.1211.00ZFP1985.2663.80ZFP4541.9041.31ZFP2073.7660 .61ZFP4633.0434.95ZFP2170.7939.75ZFPs141.5330.42ZFP2235.7513.05ZFPs344.1626.72ZFP2339.3013.34ZFPs5106 .8172.67ZFP2437.7916.89ZFPs7104.13111.79ZFP2527.469.30ZFPs994.73102.46ZFP2633.4712.39ZFPs1216.2116.60

[0842] Example 4: Determining the transcriptional regulation efficiency of the PMP22 gene by ZFP by analyzing the PMP22 transcript at the cellular level sNF02.2 cell line was incubated at 1 x 10 5Cells were mixed with 1 μg of plasmid (ZFP) and electroporated using a Neon device at 1000 V, 30 ms, and 2 pulses. Seventy-two hours after plasmid introduction, RNA was purified, and cDNA was synthesized using reverse transcriptase. Primers were designed to distinguish between the P1 and P2 promoter transcripts of the PMP22 gene, and the PMP22 transcript was analyzed using SYBR Green and a real-time gene amplification device.

[0843] GenePrimer sequence (5' to 3')PMP22 (human)P1_Forward: 5'-GGAGGGTCTTGCCTTAACA-3'P2_Forward: 5'-GCAGCGCAGCTTTGGC-3'Reverse: 5'-GTTCTGCTCAGCGGAGTTT-3'GAPDH (human)Forward: 5'-TGCACCACCAACTGCTTAGC-3' Reverse: 5'-GGCATGGACTGTGGTCATGAG-3'

[0844] As a result, it was confirmed that the ZFP according to the present invention can specifically suppress PMP22 gene expression induced by the P1 promoter without substantially affecting PMP22 gene expression induced by the P2 promoter (Fig. 3).

[0845] Example 5: Measurement of ZFP binding to the target region of the PMP22 P1 promoter at the cellular level.

[0846] sNF02.2 cell line 1 x 10 6After mixing the cells and 10 µg of plasmid (ZFP), electroporation was performed using a Neon device under the conditions of 1000 V, 30 ms, 2 pulses. 72 hours after plasmid introduction, Cut&Run analysis was performed using a Flag antibody. Primers were designed to confirm binding to the P1 promoter of the PMP22 gene in the genome, and the degree of binding of ZFP to the P1 promoter target region of PMP22 was analyzed using SYBR Green and a real-time gene amplification device. The results are shown in Figure 4. The test group expressed sZFP12 (SEQ ID NO: 358). The control group used was the same transcriptional regulatory sequence as the experimental group, except that it did not contain ZFP. IgG was used as a negative control to confirm the specific binding of the anti-Flag antibody used to the ZFP protein. As a result, it was confirmed that the P1 promoter transcription inhibition effect of PMP22 shown by ZFP according to the present invention is caused by binding to the P1 promoter of PMP22 (Fig. 4).

[0847] Example 6: Production of Schwann cell-specific transcriptional regulatory sequences.

[0848] Schwann cell-specific transcriptional regulatory sequences were constructed using the Gibson Assembly technique. The enhancer and promoter sequences used to construct the transcriptional regulatory sequences are as follows.

[0849] Enhancer

[0850] IRE1:

[0851] GACAATGGAACTTTGTTTGGTTGCCACCTCCCCCTCCTGTAGAACAAAAAGGTCTACAGTTGC CCCTCCCTGGGGCCAGCCCACACACATAGTCTCTCTGGAATGACCTTCCTTGTGTGGGTA (SEQ ID NO: 359)

[0852] IRE2:

[0853] ACTAGCCGAGGACAATGCTCCCTTGTTCGTTGTTTCAGCGCTATGTGGGTGACACCCACAGCT AGAAG(서열번호 360)

[0854] Mod4:

[0855] GGCCAGCGCATGGCCGCGCCTCGCCGACCCCGTCCCCACCCTCCTGAGGCTCCCCTAGCACAC GGCTGCCTCTCCTGTGGCCTCTTCCCTGCCGTGGGAGGTGCCAGGGGTGAGCTATGTGGAGCAAATAAAAACTATATGTTCTGGGCACACAAAGGAGGCATTGGTGTGAGATGGGTGGGTTGATGAGCCTCGTTTGTGGGAAAGTTTTCAAGAAATCTCTGCGGGAAGCCTGGCAGGCAGCCACATGCCTCTCATAGATGCAGAATTTCTGCCATCGCAAATCCACAGGCCACACCATGGCCCTTTTTGTTTCCTTTGCCCTCCCTGGTGACCCCAAGCACAGCGCGCCGTGACACAAGTATTCCACAAACAAGGAGCGTCCTCTTTGGCCCTGGCCCACACTTCCAAATTCCATGGTCTCTGCAGGGCT(서열번호 361)

[0856] Mini Mod4:

[0857] AAATCTCTGCGGGAAGCCTGGCAGGCAGCCACATGCCTCTCATAGATGCAGAATTTCTGCCATCGCAAATCCACAGGCCACACCATGGCCCTTTTTGTTTCCTTTGCCCTCCCTGGTGACCCCAAGCACAGCGCGCCGTGACACAAGTATTCCACAAACAAGGAGCGTCCTCTTTGGCCCTGGCCCACACTTCCAAATTCCATGGTCTCTGCAGGGCT(서열번호 362)

[0858] 프로모터(Promoter)

[0859] MPZ200:

[0860] TGCTCCCTGCACTCTGCCCCCCTCCCCACCACCTCTCAACTGCACATGCCAGGCTGCAATTGG TTACTGGCTGAGGACAGCCCCCTCATGCTGGGGCCCTAGGGGATTTTAAGCAGGTTCCAGGAACCCCCCGTTCAGTTCCTGGTCCCCCACTTTCTCAACCCCACAGATGCTCCGGGCCCCTGCCCCTGCCCCAGCTA(서열번호 363)

[0861] MPZ279:

[0862] CCAGTGGACACAAAGCCCTCTGTGTATGGGGTGGTATGTGTCCCCCCACCCCTCCACCCAGAC TATACAATGCCCCTTCTGCTCCCTGCACTCTGCCCCCCTCCCCACCACCTCTCAACTGCACATGCCAGGCTGCAATTGGTTACTGGCTGAGGACAGCCCCCTCATGCTGGGGCCCTAGGGGATTTTAAGCAGGTTCCAGGAACCCCCCGTTCAGTTCCTGGTCCCCCACTTTCTCAACCCCACAGATGCTCCGGGCCCCTGCCCCTGCCCCAGCTA(서열번호 364)

[0863] MPZ430:

[0864] CTCTCAGGCAAGGAGGCTGAGGAGAAATCCCTTTTTATGGCCTTTAAATTGAGGTTCCATATC TATCCCTCAGAGAAGTGTGTCTGTGTCCCTGTTTTTGTCCCTCTCCCTCACCACCCCCCACAACATTCCAGCCTGGGGCAGGGGGAGGCCAGTGGACACAAAGCCCTCTGTGTATGGGGTGGTATGTGTCCCCCCACCCCTCCACCCAGACTATACAATGCCCCTTCTGCTCCCTGCACTCTGCCCCCCTCCCCACCACCTCTCAACTGCACATGCCAGGCTGCAATTGGTTACTGGCTGAGGACAGCCCCCTCATGCTGGGGCCCTAGGGGATTTTAAGCAGGTTCCAGGAACCCCCCGTTCAGTTCCTGGTCCCCCACTTTCTCAACCCCACAGATGCTCCGGGCCCCTGCCCCTGCCCCAGCTA(서열번호 365)

[0865] MBP300:

[0866] TTTGCACAGGCCCGTATTCATATCTCATTGTTGTTTGCAGGAGAGGCAGATGCGAACCAGAAC AATGGGACCTCCTCTCAGGACACAGCGGTGACTGACTCCAAGCGCACAGCGGACCCGAAGAATGCCTGGCAGGATGCCCACCCAGCTGACCCAGGGAGCCGCCCCCACTTGATCCGCCTCTTTTCCCGAGATGCCCCGGGGAGGGAGGACAACACCTTCAAAGACAGGCCCTCTGAGTCCGACGAGCTCCAGACCATCCAAGAAGACAGTGCAGCCACCTCCGAGAGCCTG GATGTG(서열번호 366)

[0867] 제작된 슈반 세포 전사 조절 서열(인핸서 및 프로모터 포함)

[0868] IRE1 MPZ200:

[0869] GACAATGGAACTTTGTTTGGTTGCCACCTCCCCCTCCTGTAGAACAAAAAGGTCTACAGTTGCCCCTCCCTGGGGCCAGCCCACACACATAGTCTCTCTGGAATGACCTTCCTTGTGTGGGTATGCTCCCTGCACTCTGCCCCCCTCCCCACCACCTCTCAACTGCACATGCCAGGCTGCAATTGGTTACTGGCTGAGGACAGCCCCCTCATGCTGGGGCCCTAGGGGATTTTAAGCAGGTTCCAGGAACCCCCCGTTCAGTTCCTGGTCCCCCACTTTCTCAACCCCACAGATGCTCCGGGCCCCTGCCCCTGCCCCAGCTA(서열번호 367)

[0870] IRE1 MPZ430:

[0871] GACAATGGAACTTTGTTTGGTTGCCACCTCCCCCTCCTGTAGAACAAAAAGGTCTACAGTTGC CCCTCCCTGGGGCCAGCCCACACACATAGTCTCTCTGGAATGACCTTCCTTGTGTGGGTACTCTCAGGCAAGGAGGCTGAGGAGAAATCCCTTTTTATGGCCTTTAAATTGAGGTTCCATATCTATCCCTCAGAGAAGTGTGTCTGTGTCCCTGTTTTTGTCCCTCTCCCTCACCACCCCCCACAACATTCCAGCCTGGGGCAGGGGGAGGCCAGTGGACACAAAGCCCTCTGTGTATGGGGTGGTATGTGTCCCCCCACCCCTCCACCCAGACTATACAATGCCCCTTCTGCTCCCTGCACTCTGCCCCCCTCCCCACCACCTCTCAACTGCACATGCCAGGCTGCAATTGGTTACTGGCTGAGGACAGCCCCCTCATGCTGGGGCCCTAGGGGATTTTAAGCAGGTTCCAGGAACCCCCCGTTCAGTTCCTGGTCCCCCACTTTCTCAACCCCACAGATG CTCCGGGCCCCTGCCCCTGCCCCAGCTA(서열번호 368)

[0872] IRE2 IRE1 MPZ279:

[0873] ACTAGCCGAGGACAATGCTCCCTTGTTCGTTGTTTCAGCGCTATGTGGGTGACACCCACAGCT AGAAGGACAATGGAACTTTGTTTGGTTGCCACCTCCCCCTCCTGTAGAACAAAAAGGTCTACAGTTGCCCCTCCCTGGGGCCAGCCCACACACATAGTCTCTCTGGAATGACCTTCCTTGTGTGGGTACCAGTGGACACAAAGCCCTCTGTGTATGGGGTGGTATGTGTCCCCCCACCCCTCCACCCAGACTATACAATGCCCCTTCTGCTCCCTGCACTCTGCCCCCCTCCCCACCACCTCTCAACTGCACATGCCAGGCTGCAATTGGTTACTGGCTGAGGACAGCCCCCTCATGCTGGGGCCCTAGGGGATTTTAAGCAGGTTCCAGGAACCCCCCGTTCAGTTCCTGGTCCCCCACTTTCTCAACCCCACAGATGCTCCGG GCCCCTGCCCCTGCCCCAGCTA(서열번호 369)

[0874] IRE2 IRE1 MPZ430:

[0875] ACTAGCCGAGGACAATGCTCCCTTGTTCGTTGTTTCAGCGCTATGTGGGTGACACCCACAGCT AGAAGGACAATGGAACTTTGTTTGGTTGCCACCTCCCCCTCCTGTAGAACAAAAAGGTCTACAGTTGCCCCTCCCTGGGGCCAGCCCACACACATAGTCTCTCTGGAATGACCTTCCTTGTGTGGGTACTCTCAGGCAAGGAGGCTGAGGAGAAATCCCTTTTTATGGCCTTTAAATTGAGGTTCCATATCTATCCCTCAGAGAAGTGTGTCTGTGTCCCTGTTTTTGTCCCTCTCCCTCACCACCCCCCACAACATTCCAGCCTGGGGCAGGGGGAGGCCAGTGGACACAAAGCCCTCTGTGTATGGGGTGGTATGTGTCCCCCCACCCCTCCACCCAGACTATACAATGCCCCTTCTGCTCCCTGCACTCTGCCCCCCTCCCCACCACCTCTCAACTGCACATGCCAGGCTGCAATTGGTTACTGGCTGAGGACAGCCCCCTCATGCTGGGGCCCTAGGGGATTTTAAGCAGGTTCCAGGAACCCCCCGTTCAGTTCCTGGTCCCCCACTTTCTCAACCCCACAGATGCTCCGGGCCCCTGCCCCTGCCCCAGCTA(서열번호 370)

[0876] IRE2 Mod4 MPZ430:

[0877] ACTAGCCGAGGACAATGCTCCCTTGTTCGTTGTTTCAGCGCTATGTGGGTGACACCCACAGCT AGAAGGGCCAGCGCATGGCCGCGCCTCGCCGACCCCGTCCCCACCCTCCTGAGGCTCCCCTAGCACACGGCTGCCTCTCCTGTGGCCTCTTCCCTGCCGTGGGAGGTGCCAGGGGTGAGCTATGTGGAGCAAATAAAAACTATATGTTCTGGGCACACAAAGGAGGCATTGGTGTGAGATGGGTGGGTTGATGAGCCTCGTTTGTGGGAAAGTTTTCAAGAAATCTCTGCGGGAAGCCTGGCAGGCAGCCACATGCCTCTCATAGATGCAGAATTTCTGCCATCGCAAATCCACAGGCCACACCATGGCCCTTTTTGTTTCCTTTGCCCTCCCTGGTGACCCCAAGCACAGCGCGCCGTGACACAAGTATTCCACAAACAAGGAG CGTCCTCTTTGGCCCTGGCCCACACTTCCAAATTCCATGGTCTCTGCAGGGCTCTCTCAGGCAAGGAGGCTGAGGAGAAATCCCTTTTTATGGCCTTTAAATTGAGGTTCCATATCTATCCCTCAGAGAAGTGTGTCTGTGTCCCTGTTTTTGTCCCTCTCCCTCACCACCCCCCACAACATTCCAGCCTGGGGCAGGGGGAGGCCAGTGGACACAAAGCCCTCTGTGTATGGGGTGGTATGTGTCCCCCCACCCCTCCACCCAGACTATACAATGCCCCTTCTGCTCCCTGCACTCTGCCCCCCTCCCCACCACCTCTCAACTGCACATGCCAGGCTGCAATTGGTTACTGGCTGAGGACAGCCCCCTCATGCTGGGGCCCTAGGGGATTTTAAGCAGGTTCCAGGAACCCCCCGTTCAGTTCCTGGTCCCCCACTTTCTCAACCCCACAGATGCTCCGGG CCCCTGCCCCTGCCCCAGCTA(서열번호 371)

[0878] IRE2 Mod4 MBP300:

[0879] ACTAGCCGAGGACAATGCTCCCTTGTTCGTTGTTTCAGCGCTATGTGGGTGACACCCACAGCT AGAAGGGCCAGCGCATGGCCGCGCCTCGCCGACCCCGTCCCCACCCTCCTGAGGCTCCCCTAGCACACGGCTGCCTCTCCTGTGGCCTCTTCCCTGCCGTGGGAGGTGCCAGGGGTGAGCTATGTGGAGCAAATAAAAACTATATGTTCTGGGCACACAAAGGAGGCATTGGTGTGAGATGGGTGGGTTGATGAGCCTCGTTTGTGGGAAAGTTTTCAAGAAATCTCTGCGGGAAGCCTGGCAGGCAGCCACATGCCTCTCATAGATGCAGAATTTCTGCCATCGCAAATCCACAGGCCACACCATGGCCCTTTTTGTTTCCTTTGCCCTCCCTGGTGACCCCAAGCACAGCGCGCCGTGACACAAGTATTCCACAAACAAGGAG CGTCCTCTTTGGCCCTGGCCCACACTTCCAAATTCCATGGTCTCTGCAGGGCTTTTGCACAGGCCCGTATTCATATCTCATTGTTGTTTGCAGGAGAGGCAGATGCGAACCAGAACAATGGGACCTCCTCTCAGGACACAGCGGTGACTGACTCCAAGCGCACAGCGGACCCGAAGAATGCCTGGCAGGATGCCCACCCAGCTGACCCAGGGAGCCGCCCCCACTTGATCCGCCTCTTTTCCCGAGATGCCCCGGGGAGGGAGGACAACACCTTCAAAGACAGGCCCTCTGAGTCCGACGAGCTCCAG ACCATCCAAGAAGACAGTGCAGCCACCTCCGAGAGCCTGGATGTG(서열번호 372)

[0880] IRE2 Mini Mod4 MPZ279:

[0881] ACTAGCCGAGGACAATGCTCCCTTGTTCGTTGTTTCAGCGCTATGTGGGTGACACCCACAGCT AGAAGAAATCTCTGCGGGAAGCCTGGCAGGCAGCCACATGCCTCTCATAGATGCAGAATTTCTGCCATCGCAAATCCACAGGCCACACCATGGCCCTTTTTGTTTCCTTTGCCCTCCCTGGTGACCCCAAGCACAGCGCGCCGTGACACAAGTATTCCACAAACAAGGAGCGTCCTCTTTGGCCCTGGCCCACACTTCCAAATTCCATGGTCTCTGCAGGGCTCCAGTGGACACAAAGCCCTCTGTGTATGGGGTGGTATGTGTCCCCCCACCCCTCCACCCAGACTATACAATGCCCCTTCTGCTCCCTGCACTCTGCCCCCCTCCCCACCACCTCTCAACTGCACATGCCAGGCTGCAATTGGTTACTGGCTGAGGACAGCCC CCTCATGCTGGGGCCCTAGGGGATTTTAAGCAGGTTCCAGGAACCCCCCGTTCAGTTCCTGGTCCCCCACTTTCTCA ACCCCACAGATGCTCCGGGCCCCTGCCCCTGCCCCAGCTA(서열번호 373)

[0882] IRE2 Mini Mod4 MBP300:

[0883] ACTAGCCGAGGACAATGCTCCCTTGTTCGTTGTTTCAGCGCTATGTGGGTGACACCCACAGCT AGAAGAAATCTCTGCGGGAAGCCTGGCAGGCAGCCACATGCCTCTCATAGATGCAGAATTTCTGCCATCGCAAATCCACAGGCCACACCATGGCCCTTTTTGTTTCCTTTGCCCTCCCTGGTGACCCCAAGCACAGCGCGCCGTGACACAAGTATTCCACAAACAAGGAGCGTCCTCTTTGGCCCTGGCCCACACTTCCAAATTCCATGGTCTCTGCAGGGCTTTTGCACAGGCCCGTATTCATATCTCATTGTTGTTTGCAGGAGAGGCAGATGCGAACCAGAACAATGGGACCTCCTCTCAGGACACAGCGGTGACTGACTCCAAGCGCACAGCGGACCCGAAGAATGCCTGGCAGGATGCCCACCCAGCTGACCCAGGGAGCCGCCCCCACTTGATCCGCCTCTTTTCCCGAGATGCCCCGGGGAGGGAGGACAACACCTTCAAAGACAGGCCCTCTGAGTCCGACGAGCTCCAGACCATCCAAGAAGACAGTGCAGCCACCTCCGAGAGCCTGGATGTG(서열번호 374)

[0884] rMPZ IE200:

[0885] GACAATGAGACTTTGTTTGGTCGCCTCCTCCTAAAGAACAGAAAAGTCTATAATTGTTCCTCCCCAGAGCCAGCCCACACACATAGACTGCCTGGGATGACTCTCCCTGTGTGGGCGCAGAGTATACAATGCCCCTTCTGCTCCATGCCCCTGCCACCCTCCCCACCACCTCTCAATTGCACATGCCAGGCTGCAATTGGTCACTGGCTCAGGACAGCCCCCTCATGCTGGGGATCCAGGGGATTTTAAGCAGGTTCCAGAAAACACCACTCAGTTCCTTGTCCCCCGCTCTCTCCACCCCACAGACGCTCTGGGCC(서열번호 375)

[0886] hMPZ IE200:

[0887] GACAATGGAACTTTGTTTGGTTGCCACCTCCCCCTCCTGTAGAACAAAAAGGTCTACAGTTGCCCCTCCCTGGGGCCAGCCCACACACATAGTCTCTCTGGAATGACCTTCCTTGTGTGGGTACCCCTCCACCCAGACTATACAATGCCCCTTTCTGCTCCCTGC ACTCTGCCCCCCTCCCCACCACCTCTCAACTGCACATGCCAGGCTGCAATTGGTTACTGGCTGAGGACAGCCCCCTCATGCTGGGGCCCTAGGGGATTTTAAGCAGGTTCCAGGAACCCCCCGTTCAGTTCCTGGTCCCCCACTTTCTCAACCCCACAG (SEQ ID NO: 376)

[0888] Example 7: Measurement of transcriptional regulatory activity of manufactured transcriptional regulatory sequences

[0889] To measure Schwann cell transcriptional regulatory activity by flow cytometry, 3 x 10 5The dogs were mixed with 3 μg of EGFP expression plasmids containing transcriptional regulatory sequences (IRE1 MPZ200, IRE1 MPZ430, IRE2 IRE1 MPZ279, IRE2 IRE1 MPZ430, IRE2 Mod4 MPZ430, IRE2 Mod4 MBP300, IRE2 Mini Mod4 MPZ279, IRE2 Mini Mod4 MBP300) constructed in Example 6. These were electroporated using a Neon device under the conditions of 1350 V, 30 ms, 1 pulse. Seventy-two hours after plasmid introduction, the number of EGFP-expressing cells was measured using a flow cytometer (FACS AriaII). The relative transcriptional regulatory activity was calculated as the ratio of (number of Schwann cell promoter EGFP expressing cells / total number of cells) / (number of CMV promoter EGFP expressing cells / total number of cells), using the CMV promoter as a control group and rMPZ IE200 (toolgen) (SEQ ID NO: 375) and hMPZ IE200 (toolgen) (SEQ ID NO: 376) as comparison groups, and the results are shown in Fig. 5.

[0890] The seven Schwann cell transcriptional regulatory sequences according to the present invention, excluding IRE1 MPZ200, showed an activity of at least 21.7% to 68.2%, which is at least about 20% higher and at most about 380% higher than the promoters of the known ToolGen company, and in particular, it was confirmed that the activities of IRE2 Mod4 MPZ430 and IRE2 Mod4 MBP300 were excellent (Fig. 5).

[0891] Example 8: Construction of a ZFP expression vector based on Schwann cell transcriptional regulatory sequences for regulating PMP22 P1 transcriptional expression.

[0892] As a result of the experiment of Example 7 above, a polynucleotide (IRE2 Mod4 MPZ430) of a transcriptional regulatory sequence with excellent transcriptional regulatory activity was synthesized, and then an expression vector was constructed using the Gibson Assembly technique using a plasmid containing a ZFP gene targeting the PMP22 P1 promoter. The expression of ZFP (PMP22 P1 target) using the transcriptional regulatory sequence was measured by protein immunoblotting (western blot). S16 cell line 1 x 10 5 Dogs were mixed with 2 μg of ZFP expression plasmid containing the Schwann cell transcriptional regulatory sequence (IRE2 Mod4 MPZ430) produced in Example 6, and electroporated using a Neon device under the conditions of 1350 V, 30 ms, and 1 pulse. Seventy-two hours after the introduction of the plasmid, proteins were extracted. The proteins were separated by protein size using SDS-PAGE gel electrophoresis and transferred to a blot membrane. After inducing binding to the target protein using primary antibodies (flag and beta-actin), detection was performed by measuring the luminescence using an enzyme conjugated to a secondary antibody. sZFP12 (SEQ ID NO: 358), ZFP31 (SEQ ID NO: 337), and ZFP44 (SEQ ID NO: 350) were expressed using the same transcriptional regulatory sequence, respectively. The control group used the same transcriptional regulatory sequence as the experimental group, except that it did not contain ZFP. As a result, it was confirmed that all were expressed effectively (Fig. 6).

[0893] Example 9: Measurement of transcriptional regulation efficiency of the PMP22 gene through expression of ZFP (PMP22 P1 target) using transcriptional regulatory sequences.

[0894] SNU-466 cell line 1 x 10 5The cells were mixed with 1 μg of ZFP expression plasmids containing Schwann cell transcription regulatory sequences (IRE2 Mod4 MPZ430, IRE2 Mod4 MBP300) produced in Example 6, and electroporation was performed using a Neon device under the conditions of 1000 V, 30 ms, 1 pulse. Seventy-two hours after the introduction of the plasmids, RNA was purified and cDNA was synthesized using reverse transcriptase. Primers were designed to distinguish between the P1 promoter transcript and the P2 promoter transcript of the PMP22 gene, and the PMP22 transcript was analyzed using SYBR Green and a real-time gene amplification device, and the results are shown in Fig. 7. For each transcription regulatory sequence tested, sZFP12 (SEQ ID NO: 358), ZFP31 (SEQ ID NO: 337), and ZFP44 (SEQ ID NO: 350) were expressed. The control group used had the same transcriptional regulatory sequence as the experimental group, except that it did not contain ZFP.

[0895] As a result, both IRE2 Mod4 MPZ430 and IRE2 Mod4 MBP300 according to the present invention showed lower transcription efficiency of the P1 promoter of the PMP22 gene than the control group, and in particular, when ZFP44 was used, the transcription efficiency was suppressed by 42% and 52%, respectively, and the transcription efficiency was also low when sZFP12 and ZFP31 were used. In addition, when IRE2 Mod4 MPZ430 and IRE2 Mod4 MBP300 according to the present invention were used as transcription regulatory sequences, it was confirmed that both specifically suppressed the expression of the PMP 22 protein by the P1 promoter compared to the P2 promoter of the PMP22 gene (Fig. 7).

Claims

1. An isolated zinc finger protein comprising at least three zinc fingers and binding to the promoter or a part thereof of the PMP22 gene in a nervous system cell to reduce the expression of the PMP22 protein.

2. An isolated zinc finger protein according to claim 1, wherein the promoter is a P1 promoter.

3. An isolated zinc finger protein that specifically inhibits the expression of PMP 22 protein by the P1 promoter compared to the P2 promoter of the PMP22 gene in the first paragraph.

4. An isolated zinc finger protein according to any one of claims 1 to 3, which binds to a nucleic acid sequence portion selected from the group consisting of SEQ ID NOs: 3 to 48 and the following sequences or a complementary sequence thereof. (1) 5'-CAGGGAGCA-3' (2) 5'-CACCAGGGA-3' (3) 5'-GGAGCACCA-3' (4) 5'-CAGGGAACA-3' (5) 5'-GGTGGTGCT-3' (6) 5'-CCTGTAACT-3' (7) 5'-CCTGTAACT-3' (8) 5'-AAGCCAGAC-3' (9) 5'-GTAACTGAA-3' (10) 5'-CCAGACCAG-3' (11) 5'-ACTGAAGCC-3' (12) 5'-GACCAGGCG-3' 5. An isolated zinc finger protein comprising a set of amino acid sequences selected from the group consisting of (1) to (52) according to any one of claims 1 to 4: (1) Sequence numbers 49 to 52; (2) Sequence numbers 53 to 56; (3) Sequence numbers 57 to 60; (4) Sequence numbers 61 to 64; (5) Sequence numbers 65 to 68; (6) Sequence numbers 69 to 72; (7) Sequence numbers 73 to 76; (8) Sequence numbers 77 to 80; (9) Sequence numbers 81 to 84; (10) Sequence numbers 85 to 88; (11) Sequence numbers 89 to 92; (12) Sequence numbers 93 to 96; (13) Sequence numbers 97 to 100; (14) Sequence numbers 101 to 104; (15) Sequence numbers 105 to 108; (16) Sequence numbers 109 to 112; (17) Sequence numbers 113 to 116; (18) Sequence numbers 117 to 120; (19) Sequence numbers 121 to 124; (20) Sequence numbers 125 to 129; (21) Sequence numbers 130 to 134; (22) Sequence numbers 135 to 139; (23) Sequence numbers 140 to 144; (24) Sequence numbers 145 to 149; (25) Sequence numbers 150 to 154; (26) Sequence numbers 155 to 159; (27) Sequence numbers 160 to 164; (28) Sequence numbers 165 to 169; (29) Sequence numbers 170 to 174; (30) Sequence numbers 175 to 179; (31) Sequence numbers 180 to 184; (32) Sequence numbers 185 to 189; (33) Sequence numbers 190 to 194; (34) Sequence numbers 195 to 199; (35) Sequence numbers 200 to 204; (36) Sequence numbers 205 to 210; (37) Sequence numbers 211 to 216; (38) Sequence numbers 217 to 222; (39) Sequence numbers 223 to 228; (40) Sequence numbers 229 to 234; (41) Sequence numbers 235 to 240; (42) Sequence numbers 241 to 246; (43) Sequence numbers 247 to 252; (44) Sequence numbers 253 to 258; (45) Sequence numbers 259 to 264; (46) Sequence numbers 265 to 270; (47) Sequence numbers 271 to 276; (48) Sequence numbers 277 to 282; (49) Sequence numbers 283 to 288; (50) Sequence numbers 289 to 294; (51) Sequence numbers 295 to 300; and (52) Sequence numbers 301 to 306.

6. An isolated zinc finger protein comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 307 to 358, according to any one of claims 1 to 5.

7. An isolated zinc finger protein according to any one of claims 1 to 6, which reduces the expression of PMP22 protein by the P1 promoter in Schwann cells of Charcot-Marie-Tooth patients by 20% or more, 30% or more, or 40% or more compared to normal individuals.

8. An isolated polynucleotide encoding an isolated zinc finger protein according to any one of claims 1 to 7.

9. A polynucleotide according to claim 8, further comprising a Schwann cell-specific promoter operably linked to the polynucleotide.

10. A polynucleotide according to claim 9, wherein the Schwann cell-specific promoter is an MPZ promoter, an MBP promoter, a PMP22 promoter, or a MAG promoter.

11. A polynucleotide according to any one of claims 8 to 10, further comprising a transcription repression domain.

12. A polynucleotide according to claim 11, wherein the transcription repression domain is a KRAB domain.

13. A polynucleotide according to any one of claims 8 to 10, which does not additionally comprise a transcription repression domain.

14. A polynucleotide according to claim 13, wherein the transcription repression domain is a KRAB domain.

15. A polynucleotide comprising a transcriptional regulatory sequence comprising a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or at least 100% sequence identity with a nucleotide sequence selected from the group consisting of SEQ ID NOs: 363 to 366.

16. A polynucleotide comprising a transcriptional regulatory sequence comprising a nucleotide sequence having a sequence identity of at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or at least 100% with the nucleotide sequence of SEQ ID NO: 365 or 366 in accordance with paragraph 15.

17. A polynucleotide according to claim 15 or 16, wherein the transcription regulatory sequence further comprises one or more enhancer sequences.

18. A polynucleotide according to claim 17, wherein one or more enhancers each independently have a sequence identity of 80% or more, 85% or more, 90% or more, 95% or more, 99% or more, or 100% or more with a nucleotide sequence selected from the group consisting of SEQ ID NOs: 359 to 362.

19. A polynucleotide according to claim 15, wherein the transcription regulatory sequence comprises a nucleotide sequence having a sequence identity of 80% or more, 85% or more, 90% or more, 95% or more, 99% or more, or 100% or more with a nucleotide sequence selected from the group consisting of SEQ ID NOs: 367 to 374.

20. A polynucleotide according to any one of claims 15 to 19, further comprising a target gene sequence, wherein the target gene is a nucleic acid encoding a gene to be expressed in a Schwann cell or a protein to be expressed in a Schwann cell, and is operably linked to a transcriptional regulatory sequence.

21. A polynucleotide according to claim 20, wherein the target gene is a nucleic acid encoding a zinc finger protein that binds to the promoter of the PMP22 gene or a part thereof in a nervous system cell and reduces the expression of the PMP22 protein.

22. A polynucleotide according to claim 21, wherein the zinc finger protein specifically inhibits the expression of the PMP 22 protein by the P1 promoter compared to the P2 promoter of the PMP22 gene.

23. A polynucleotide according to claim 21 or 22, further comprising a transcription repression domain.

24. A polynucleotide according to claim 23, wherein the transcription repression domain is a KRAB domain.

25. A polynucleotide according to claim 21 or 22, which does not additionally comprise a transcription repression domain.

26. A polynucleotide according to claim 25, wherein the transcription repression domain is a KRAB domain.

27. A polynucleotide according to any one of claims 21 to 26, wherein the zinc finger protein binds to a nucleic acid sequence portion selected from the group consisting of SEQ ID NOs: 3 to 48 and the following sequences or a complementary sequence thereof. (1) 5'-CAGGGAGCA-3' (2) 5'-CACCAGGGA-3' (3) 5'-GGAGCACCA-3' (4) 5'-CAGGGAACA-3' (5) 5'-GGTGGTGCT-3' (6) 5'-CCTGTAACT-3' (7) 5'-CCTGTAACT-3' (8) 5'-AAGCCAGAC-3' (9) 5'-GTAACTGAA-3' (10) 5'-CCAGACCAG-3' (11) 5'-ACTGAAGCC-3' (12) 5'-GACCAGGCG-3' 28. A polynucleotide according to any one of claims 21 to 27, wherein the zinc finger protein comprises a set of amino acid sequences selected from the group consisting of (1) to (52): (1) Sequence numbers 49 to 52; (2) Sequence numbers 53 to 56; (3) Sequence numbers 57 to 60; (4) Sequence numbers 61 to 64; (5) Sequence numbers 65 to 68; (6) Sequence numbers 69 to 72; (7) Sequence numbers 73 to 76; (8) Sequence numbers 77 to 80; (9) Sequence numbers 81 to 84; (10) Sequence numbers 85 to 88; (11) Sequence numbers 89 to 92; (12) Sequence numbers 93 to 96; (13) Sequence numbers 97 to 100; (14) Sequence numbers 101 to 104; (15) Sequence numbers 105 to 108; (16) Sequence numbers 109 to 112; (17) Sequence numbers 113 to 116; (18) Sequence numbers 117 to 120; (19) Sequence numbers 121 to 124; (20) Sequence numbers 125 to 129; (21) Sequence numbers 130 to 134; (22) Sequence numbers 135 to 139; (23) Sequence numbers 140 to 144; (24) Sequence numbers 145 to 149; (25) Sequence numbers 150 to 154; (26) Sequence numbers 155 to 159; (27) Sequence numbers 160 to 164; (28) Sequence numbers 165 to 169; (29) Sequence numbers 170 to 174; (30) Sequence numbers 175 to 179; (31) Sequence numbers 180 to 184; (32) Sequence numbers 185 to 189; (33) Sequence numbers 190 to 194; (34) Sequence numbers 195 to 199; (35) Sequence numbers 200 to 204; (36) Sequence numbers 205 to 210; (37) Sequence numbers 211 to 216; (38) Sequence numbers 217 to 222; (39) Sequence numbers 223 to 228; (40) Sequence numbers 229 to 234; (41) Sequence numbers 235 to 240; (42) Sequence numbers 241 to 246; (43) Sequence numbers 247 to 252; (44) Sequence numbers 253 to 258; (45) Sequence numbers 259 to 264; (46) Sequence numbers 265 to 270; (47) Sequence numbers 271 to 276; (48) Sequence numbers 277 to 282; (49) Sequence numbers 283 to 288; (50) Sequence numbers 289 to 294; (51) Sequence numbers 295 to 300; and (52) Sequence numbers 301 to 306.

29. A polynucleotide according to claim 28, wherein the zinc finger protein comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 307 to 358.

30. A polynucleotide according to any one of claims 21 to 29, which reduces the expression of PMP22 protein by 20% or more, 30% or more, or 40% or more when expressed in Schwann cells of a Charcot-Marie-Tooth patient.

31. A delivery vehicle comprising a polynucleotide according to any one of claims 8 to 30.

32. A vector according to claim 31, which is a plasmid vector, a viral vector or a non-viral vector.

33. A vector according to claim 32, which is a viral vector selected from the group consisting of retrovirus, adenovirus, lentivirus, poxvirus, vaccinia virus, herpesvirus, and adeno-associated virus vector (AAV).

34. A delivery vehicle in claim 32, which is a non-viral vector selected from the group consisting of liposomes, exosomes, lipid nanoparticles, polymer nanoparticles, extracellular vesicles, synthetic vesicles, and virus-like particles.

35. A pharmaceutical composition for treating a disease associated with Schwann cells, comprising a zinc finger protein according to any one of claims 1 to 7, a polynucleotide according to any one of claims 8 to 30, or a transporter according to any one of claims 31 to 34.

36. A pharmaceutical composition according to claim 35, wherein the disease associated with Schwann cells is Charcot-Marie-Tooth disease.

37. A pharmaceutical composition according to claim 36, which, when administered to a Charcot-Marie-Tooth patient, reduces the expression of PMP22 protein by the P1 promoter in Schwann cells by 20% or more, 30% or more, or 40% or more compared to a normal person.

38. A method for treating a disease associated with Schwann cells, comprising administering to a patient in need of treatment for Charcot-Marie-Tooth disease a zinc finger protein according to any one of claims 1 to 7, a polynucleotide according to any one of claims 8 to 30, or a carrier according to any one of claims 31 to 34.

39. A method of treating, in claim 38, a disease associated with Schwann cells being Charcot-Marie-Tooth disease.

40. A method of treating, in claim 39, wherein the expression of PMP22 protein by the P1 promoter in Schwann cells of a Charcot-Marie-Tooth patient is reduced by 20% or more, 30% or more, or 40% or more compared to a normal person.

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