RU2788605C2 - OLIGODENDROCYTE-SPECIFIC PROMOTOR, microRNA SPECIFIC TO PLP1 GENE, VECTOR CONTAINING SPECIFIED PROMOTOR AND/OR SPECIFIED microRNA, AND PHARMACEUTICAL COMPOSITION CONTAINING SPECIFIED VECTOR - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: microRNA specific for PLP1 gene, containing a pair of nucleotide sequences, consisting of an antisense sequence and a sense sequence, a vector capable of suppressing expression of PLP1 gene, a pharmaceutical composition for the treatment of a disease related to PLP1 gene anomaly, and an inhibitor of expression of PLP1 gene are presented. A method for the treatment of a disease related to PLP1 gene anomaly and a method for suppression of expression of PLP1 gene are also presented.

EFFECT: invention allows for use it for the treatment of a disease related to PLP1 gene anomaly, for example, such as Pelizaeus-Merzbacher disease.

19 cl, 13 dwg, 3 tbl, 10 ex

Description

FIELD OF TECHNOLOGY

[0001] The present invention relates to an oligodendrocyte-specific promoter, in particular, to an oligodendrocyte-specific promoter containing a nucleic acid at least 90% identical to the nucleotide sequence described in SEQ ID NO: 1. The present invention also relates to miRNA, specific to the PLP1 gene. In addition, the present invention relates to a vector containing the specified promoter and/or the specified siRNA, and to a pharmaceutical composition containing such a vector.

BACKGROUND OF THE INVENTION

[0002] Hypomyelin leukodystrophy is a general term for diseases of the brain in children caused by poor development of the white matter of the brain, with 11 diseases currently known. A typical disease among them is Pelizeus-Merzbacher disease (PMD). PMD is characterized by impaired myelination of the central nervous system and is a rare intractable disease that causes severe motor developmental disorders and neurological symptoms immediately after birth. The estimated incidence in Japan is 1.45 cases per 100,000 male births. Currently, the necessary means of treatment are not available.

[0003] Myelin is produced by oligodendrocytes (oligodendroglia), which is a type of glial cell present in the brain, and has a multi-layered structure of insulating phospholipids surrounding neuronal axons. The main protein of myelin is proteolipid protein (PLP), other components are also known as myelin basic protein (MBP) and human 2',3'-cyclic nucleotide phosphodiesterase (CNP). PMD is caused by abnormalities in the proteolipid protein gene (PLP1 gene). Among them, the most frequent mutation is the PLP1 duplication (approximately 60%). Duplication of PLP1 leads to overexpression of the PLP1 gene; therefore, normalization of its expression level can be expected to have a therapeutic effect.

[0004] Non-Patent Document 1 discloses adeno-associated virus (AAV) mediated expression of green fluorescent protein (GFP) driven by the myelin basic protein (MBP) promoter or glial fibrillary acidic protein (GFAP) promoter in the brain of mice during development, and describes that GFP expression driven by the GFAP promoter is highly specific for astrocytes after injection of the vector into the brains of neonatal and adult mice. Non-Patent Document 1 also states that the selectivity of the MBP promoter for oligodendrocytes was low after administration of AAV to neonatal mice, but excellent after administration of the vector on the 10th day after birth. This document proposes that administration of AAV driven by a cell-specific promoter directly into the developed postnatal brain results in targeted long-term expression of the transgene in glial cells.

[0005] Non-Patent Document 2 describes the development of an oligodendrocyte-targeted gene therapy against Peliceus-Merzbacher-like disease, wherein the GJC2/Cx47 gene is inserted downstream of the myelin basic protein promoter and administered to 10-day-old mice by a single intracerebral injection into an internal capsule using vector based adeno-associated virus (AAV.MBP.Cx47myc).

[0006] Non-Patent Documents 1 and 2 disclose gene therapy and a corresponding promoter using an AAV vector, but do not describe any gene therapy targeting PLP1 gene abnormalities and suitable promoters or miRNAs therefor.

Bibliography

Non-Patent Literature

[0007] Non-Patent Document 1: von Jonquieres G et al., Glial promoter selectivity following AAV-delivery to the immature brain. PLOS One. 2013 Jun 14; 8(6): e65646.

Non-Patent Document 2: Georgiou E et al., Gene therapy targeting oligodendrocytes provides therapeutic benefit in a leukodystrophy model. brain. 2017 Mar 1; 140(3): 599-616.

SUMMARY OF THE INVENTION

Technical task

[0008] The present invention relates to a vector capable of oligodendrocyte-specific suppression of the expression of the PLP1 gene for the treatment of PMD caused by an abnormality of the PLP1 gene, an appropriate promoter and an appropriate miRNA, and a pharmaceutical composition containing the vector.

The solution of the problem

[0009] The present inventors have found that the human phosphodiesterase 2',3'-cyclic nucleotide (CNP) promoter having the sequence shown in SEQ ID NO: 1 can drive gene expression in an oligodendrocyte-specific and highly efficient manner, and that the vector An AAV containing this promoter and a microRNA specific for the PLP1 gene downstream of and operably linked to the promoter can suppress the expression of the PLP1 gene, and thus completes the present invention.

[0010] Namely, the present invention relates to the following aspects.

[1] An oligodendrocyte-specific promoter containing a nucleic acid at least 90% identical to the nucleotide sequence described in SEQ ID NO: 1.

[2] The promoter according to [1], containing a nucleic acid having the nucleotide sequence described in SEQ ID NO: 1.

[3] Oligodendrocyte-specific vector containing the promoter of [1] or 2].

[4] The vector according to [3], further containing a microRNA sequence specific for the human PLP1 gene, operably linked to the promoter.

[5] A microRNA specific for the PLP1 gene, containing a pair of nucleotide sequences consisting of an antisense sequence and a sense sequence, where the pair of nucleotide sequences is selected from the group consisting of pairs of antisense sequences shown in the left column of Table 1 and sense sequences shown next with antisense sequences in the right column of the table.

[6] MicroRNA according to [5], containing a nucleotide sequence selected from the group consisting of SEQ ID NO: 2-7 and 24-51.

[7] MicroRNA according to [5] containing a nucleotide sequence selected from the group consisting of SEQ ID NOs: 2-5, 7, 24, 26, 29-32, 35, 36, 42 and 51.

[8] MicroRNA according to [5] containing a nucleotide sequence selected from the group consisting of SEQ ID NOs: 2, 29, 31 and 32.

[9] A vector containing a microRNA sequence according to any one of [5]-[8].

[10] The vector according to [9], where the miRNA sequence is operably linked to an oligodendrocyte-specific promoter.

[11] The vector according to [10], where the oligodendrocyte-specific promoter is the promoter according to [1] or 2].

[12] The vector according to any one of [3], [4] and [9]-[11], wherein the vector is an adeno-associated virus (AAV) vector.

[13] A pharmaceutical composition containing the vector according to any one of [3], [4] and [9]-[12].

[14] The pharmaceutical composition according to [13], which is a pharmaceutical composition for treating a disease associated with an abnormality of the PLP1 gene.

[15] The pharmaceutical composition of [14], wherein the disease is Pelizeus-Merzbacher disease, type 2 spastic paraplegia, or multiple sclerosis.

[16] The pharmaceutical composition according to [14], wherein the disease is Peliceus-Merzbacher disease.

[17] The pharmaceutical composition according to [13], which is a PLP1 gene expression inhibitor that suppresses PLP1 gene expression.

[18] A method for treating a disease associated with an abnormality of the PLP1 gene, comprising administering an effective amount of the vector according to any one of [3], [4] and [9]-[12] to a patient in need of treatment.

[19] The method of [18], wherein the disease is Pelizeus-Merzbacher disease, type 2 spastic paraplegia, or multiple sclerosis.

[20] The method according to [18], wherein the disease is Pelizeus-Merzbacher disease.

[21] A method for suppressing PLP1 gene expression, comprising administering an effective amount of the vector according to any one of [3], [4] and [9]-[12] to a patient in need of treatment.

[22] The vector according to any one of [3], [4] and [9]-[12] for use in a method for treating a disease associated with an abnormality of the PLP1 gene.

[23] The vector of [22], wherein the disease is Pelizeus-Merzbacher disease, type 2 spastic paraplegia, or multiple sclerosis.

[24] The vector of [22], wherein the disease is Pelizeus-Merzbacher disease.

Useful effects of the invention

[0011] The AAV vector containing the promoter of the present invention is capable of expressing the gene in an oligodendrocyte-specific and highly efficient manner, and furthermore, it successfully suppresses the expression of the PLP1 gene by incorporating a microRNA sequence specific for the human PLP1 gene operably linked to the promoter, and therefore, it can be used as a therapeutic agent against PMD caused by PLP1 duplication, etc.

BRIEF DESCRIPTION OF THE DRAWINGS

[0012] [Fig. 1] In FIG. 1 is a schematic diagram of the AAV vector described in Example 1.

[Fig. 2] In FIG. 2 shows photographs showing the result of the specificity analysis of AAV-expressing cells by the immunofluorescent staining method described in Example 2.

[Fig. 3] In FIG. 3 is a graph showing the results of measuring downregulation (knockdown) sites in terms of the expression level of the PLP1 gene by the fluorescence intensity generated during the PLP1 immunofluorescence staining process as described in Example 3.

[Fig. 4] In FIG. 4 shows graphs showing the results of measuring downregulation (knockdown) in terms of the expression level of the PLP1 gene by the quantitative PCR method as described in Example 4. (A) and (B) show the relative expression levels of the PLP1 gene and the relative expression levels of the Olig2 gene, respectively. .

[Fig. 5] In FIG. 5 shows photographs showing the results of histological analysis by immunostaining after administration of PLP1-Tg to mice as described in Example 5.

[Fig. 6] In FIG. 6 shows photographs representing the results of micromorphological analysis using an electron microscope after administration of PLP1-Tg to mice, as described in example 5.

[Fig. 7] In FIG. 7 is a graph demonstrating the increase in lifespan after administration of PLP1-Tg to mice as described in Example 5.

[Fig. 8] FIG. 8 is a graph showing the increase in body weight after administration of PLP1-Tg to mice as described in Example 5.

[Fig. 9] FIG. 9 is a graph showing the results of measuring the expression suppression (knockdown) in terms of the expression level of the PLP1 gene by the quantitative PCR method as described in Example 6.

[Fig. 10] FIG. 10 is a graph showing the results of measuring the expression suppression (knockdown) in terms of the expression level of the PLP1 gene by the quantitative PCR method as described in Example 7.

[Fig. 11] In FIG. 11 is a graph showing the results of measuring the expression suppression (knockdown) in terms of the expression level of the PLP1 gene by the quantitative PCR method as described in Example 8.

[Fig. 12] FIG. 12 is a graph showing the results of measuring the expression suppression (knockdown) in terms of the expression level of the PLP1 gene by the quantitative PCR method as described in Example 9.

[Fig. 13] FIG. 13 is a graph showing the results of measuring downregulation (knockdown) sites in terms of the expression level of the PLP1 gene by the fluorescence intensity generated during the PLP1 immunofluorescence staining process as described in Example 10.

DESCRIPTION OF EMBODIMENTS

[0013] [Promoter]

The promoter of the present embodiment is an oligodendrocyte-specific promoter comprising a nucleic acid whose sequence is at least 90%, preferably at least 95%, more preferably at least 98% identical to the nucleotide sequence described in SEQ ID NO : 1, most preferably said promoter contains a nucleic acid having the nucleotide sequence described in SEQ ID NO: 1. In addition to the nucleic acid, at least 90% identical to the nucleotide sequence described in SEQ ID NO: 1, the promoter may contain nucleotide sequences, cloning sites in vectors, such as restriction enzyme recognition sites, and the att sequence for gateway cloning.

[0014] The nucleic acid having the nucleotide sequence described in SEQ ID NO: 1 is a human CNP promoter that can be obtained from chr17:40118309-40120101 of the standard human genome sequence (GRCh37/hg19). The human CNP gene is a gene specifically expressed in brain oligodendrocytes, and its specificity is believed to be determined by the sequence of said promoter. The present inventors have found that the sequence of the human CNP promoter described in SEQ ID NO: 1 can provide oligodendrocyte-specific expression of genes other than the CNP gene downstream of this promoter.

[0015] The promoter of the present embodiment is a promoter that specifically functions in oligodendrocytes, whereby genes operably linked to and downstream of the promoter are specifically expressed in oligodendrocytes. The promoter of the present embodiment can be obtained using genetic engineering techniques. For example, a human CNP promoter gene is obtained from the human genome, and a promoter having a desired nucleotide sequence is obtained by deletion, insertion, or substitution of some nucleotides. Alternatively, a promoter having the desired nucleotide sequence can be obtained by partial or complete synthesis.

[0016] [miRNA]

miRNA usually contains an antisense sequence and a sense sequence and can form a hairpin structure, thereby becoming a mature miRNA. The antisense strand of the mature miRNA binds to the target mRNA causing degradation or repression of the translation of said mRNA and thereby downregulation of the expression of the target gene. The microRNA of the present embodiment is a microRNA specific for the PLP1 gene, in particular the human PLP1 gene, contains an antisense sequence and a sense sequence, and can suppress the expression of the PLP1 gene.

[0017] Preferably, the microRNA of the present embodiment contains a 5' flanking sequence, an antisense sequence, a stalk loop sequence, a sense sequence, and a 3' flanking sequence in that order, starting from the 5' end. The configuration calls for the 5' flanking sequence to be 25-100 nucleotides long, the stalk loop sequences 4-30 nucleotides long, and the 3' flanking sequence 25-100 nucleotides long.

[0018] The flanking sequence can be any sequence known to be commonly expressed in oligodendrocytes, for example, a 5' flanking sequence and a 3' flanking sequence derived from miR155 can be used. In addition to the 5' flanking sequence and 3' flanking sequence derived from miR155, the 5' flanking sequence and 3' flanking sequence of miR-138, miR-219, miR-9, miR-23a, miR-388 and miR-297c, which are known to be expressed in oligodendrocytes.

[0019] A microRNA sequence specific for the human PLP1 gene can be designed using software such as BLOCK-iT(TM) RNAi Designer (ThermoFisher Scientific, USA), for example, the human PLP1 microRNA candidate nucleotide sequences described below can be used.

Candidate nucleotide sequence of human PLP1 miRNA 1 (SEQ ID NO: 2):

5'-AAAGGAAGAAGAAAAGAGGCAGGTTTTTGGCCACTGACTGACCTGCCTCTCTTCTTCCTTT-3'

Candidate nucleotide sequence of human PLP1 miRNA 2 (SEQ ID NO: 3):

5'-AACACCAGGAGCCACACAACGGTTTTGGCCACTGACTGACCGTTGTGTCTCCTGGTGTT-3'

Candidate nucleotide sequence of human PLP1 miRNA 3 (SEQ ID NO: 4):

5'-TTCCATGGGAGAACACCATACGTTTTGGCCACTGACTGACGTATGGTGCTCCCATGGAA-3'

Candidate nucleotide sequence of human PLP1 miRNA 4 (SEQ ID NO: 5):

5'-TGAGCAGGGAAACCAGTGTAGGTTTTGGCCACTGACTGACCTACACTGTTCCCTGCTCA-3'

Candidate nucleotide sequence of human PLP1 miRNA 5 (SEQ ID NO: 6)

5'-AGGGCTTTTCTGATTGACAGCCGTTTTGGCCACTGACTGACGGCTGTCACAGAAAGCCCT-3'

Candidate nucleotide sequence of human PLP1 miRNA 6 (SEQ ID NO: 7):

5'-ACCCCAAAGAAACACAATCCAGTTTTGGCCACTGACTGACtggattgtttctttggggt-3'

Candidate nucleotide sequence of human PLP1 miRNA 7 (SEQ ID NO: 24)

5'-ACAAATGCAGCAATAAACAGGGTTTTGGCCACTGACTGACCCTGTTTAGCTGCATTTGT-3'

Candidate nucleotide sequence of human PLP1 miRNA 8 (SEQ ID NO: 25):

5'-AATAGACTGGCAGGTGGTCCAGTTTTGGCCACTGACTGACTGGACCACCGCCAGTCTATT-3'

Candidate nucleotide sequence of human PLP1 miRNA 9 (SEQ ID NO: 26)

5'-AAAAGAATGAGCTTGATGTTGGGTTTTGGCCACTGACTGACCCAACATCGCTCATTCTTT-3'

Candidate nucleotide sequence of human PLP1 miRNA 10 (SEQ ID NO: 27):

5'-AGATACTCATAGTCTTGGTAGGTTTTGGCCACTGACTGACCTACCAAGTATGAGTATCT-3'

Candidate nucleotide sequence of human PLP1 miRNA 11 (SEQ ID NO: 28):

5'-AAGCCCATGTCTTTGGGACTCGTTTTGGCCACTGACTGACGAGTCCCAGACATGGGCTT-3'

[0020] Alternatively, to construct a miRNA sequence specific for the human PLP1 gene, artificial miRNAs are constructed as described in the Examples section and the optimal miRNA sequence is selected, which is then used for miRNA synthesis.

[0021] The miRNA of the present embodiment comprises a nucleotide sequence pair consisting of an antisense sequence and a sense sequence, where the nucleotide sequence pair may be selected from the group consisting of pairs containing the antisense sequence described in the left column of Table 1 and the sense sequence, listed to the right of the antisense sequence in the right column of the table.

Table 1. Antisense and sense miRNA sequences designed for the human PLP1 gene

Antisense sequence semantic sequence AAAGGAAGAAGAAAAGAGGCAG（SEQ ID NO: 52） CTGCCTCTCTTCTTCCTTT（SEQ ID NO: 53） AACACCAGGAGCCACACAACG（SEQ ID NO: 54） CGTTGTGTCTCCTGGTGTT（SEQ ID NO: 55） TTCCATGGGAGAACACCATAC（SEQ ID NO: 56） GTATGGTGCTCCCATGGAA（SEQ ID NO: 57） TGAGCAGGGAAACCAGTGTAG（SEQ ID NO: 58） CTACACTGTTCCCTGCTCA（SEQ ID NO: 59） AGGGCTTTCTGATTGACAGCC（SEQ ID NO: 60） GGCTGTCACAGAAAGCCCT（SEQ ID NO: 61） ACCCCAAAGAAACACAATCCA（SEQ ID NO: 62） TGGATTGTTTCTTTGGGGT（SEQ ID NO: 63） ACAAATGCAGCAATAAACAGG（SEQ ID NO: 64） CCTGTTTAGCTGCATTTGT（SEQ ID NO: 65） AATAGACTGGCAGGTGGTCCA（SEQ ID NO: 66） TGGACCACCGCCAGTCTATT（SEQ ID NO: 67） AAAGAATGAGCTTGATGTTGG（SEQ ID NO: 68） CCAACATCGCTCATTCTTT（SEQ ID NO: 69） AGATACTCATAGTCTTGGTAG（SEQ ID NO: 70） CTACCAAGTATGAGTATCT（SEQ ID NO: 71） AGAAACACAATCCAGTGGCCA（SEQ ID NO: 72） TGGCCACTATTGTGTTTCT（SEQ ID NO: 73） AAATAGGTCTCAATTAGCTTT（SEQ ID NO: 74） AAAGCTAAGAGACCTATTT（SEQ ID NO: 75） AAGAAACACAATCCAGTGGCC（SEQ ID NO: 76） GGCCACTGTTGTGTTTCTT（SEQ ID NO: 77） TAAACAGGTGGAAGGTCATTT（SEQ ID NO: 78） AAATGACCCCACCTGTTTA（SEQ ID NO: 79） TTGTAGTCGCCAAAAGATCTGC（SEQ ID NO: 80） GCAGATTCGGCGACTACAA（SEQ ID NO: 81） AATTAGAGCCTCCATTCCTTT（SEQ ID NO: 82） AAAGGAATAGGCTCTAATT（SEQ ID NO: 83） TTAAGGACGGCAAAGTTGTAA（SEQ ID NO: 84） TTACAACTGCCGTCCTTAA（SEQ ID NO: 85） TTTAAGGACGGCAAAGTTGTA（SEQ ID NO: 86） TACAACTTCCGTCCTTAAA（SEQ ID NO: 87） ATGTCTTTGGGACTCTGACTC（SEQ ID NO: 88） GAGTCAGACCCCAAAGACAT（SEQ ID NO: 89） TATCTATCCTGTGTCTACCAG（SEQ ID NO: 90） CTGGTAGACAGGATAGATA（SEQ ID NO: 91） AAATTACTTTCTGATTCCTCAG（SEQ ID NO: 92） CTGAGGATGAAAGTAATTT（SEQ ID NO: 93） TCTAACAAGCCCATGTCTTTG（SEQ ID NO: 94） CAAAGACAGGCTTGTTAGA（SEQ ID NO: 95） AATTACTTTCTGATTCCTCAGG（SEQ ID NO: 96） CCTGAGGAAGAAAGTAATT（SEQ ID NO: 97） AGTAAATGTACACAGGCACAG（SEQ ID NO: 98） CTGTGCCTGTACATTTACT（SEQ ID NO: 99） TAAGTAAGGTTGGCTGAGTTA（SEQ ID NO: 100） TAACTCAGAACCTTACTTA（SEQ ID NO: 101） TTTCTGTGGGTGAAAGATCCTT（SEQ ID NO: 102） AAGGATCTCACCCACAGAA（SEQ ID NO: 103） AGAAGATGCTGACAACACCCT（SEQ ID NO: 104） AGGGTGTTCAGCATCTTCT（SEQ ID NO: 105） AATTGTAGCCGGCTGGCTAGT（SEQ ID NO: 106） ACTAGCCACGGCTACAATT（SEQ ID NO: 107） AGATTTTGGGCAAACCGCTCTTA（SEQ ID NO: 108） TAAGAGCGTGCCCAAAATCT（SEQ ID NO: 109） ATTCTACGCTCCCTTATGCTG（SEQ ID NO: 110） CAGCATAAGAGCGTAGAAT（SEQ ID NO: 111） TGGTAATAGAGAGACCAGAAT（SEQ ID NO: 112） ATTCTGGTCTCTATTACCA（SEQ ID NO: 113） TATAGATGGCAAGAGGACCAA（SEQ ID NO: 114） TTGGTCCTTGCCATCTATA（SEQ ID NO: 115） AAACCAGTGTAGCTGCAGCCC（SEQ ID NO: 116） GGGCTGCATACACTGGTTT（SEQ ID NO: 117）

[0022] The microRNA of the present embodiment preferably contains one of the following pairs of nucleotide sequences.

- A pair of nucleotide sequences consisting of SEQ ID NO: 52 as the antisense sequence and SEQ ID NO: 53 as the sense sequence.

- A pair of nucleotide sequences consisting of SEQ ID NO: 54 as the antisense sequence and SEQ ID NO: 55 as the sense sequence.

- A pair of nucleotide sequences consisting of SEQ ID NO: 56 as the antisense sequence and SEQ ID NO: 57 as the sense sequence.

- A pair of nucleotide sequences consisting of SEQ ID NO: 58 as the antisense sequence and SEQ ID NO: 59 as the sense sequence.

- A pair of nucleotide sequences consisting of SEQ ID NO: 62 as the antisense sequence and SEQ ID NO: 63 as the sense sequence.

- A pair of nucleotide sequences consisting of SEQ ID NO: 64 as the antisense sequence and SEQ ID NO: 65 as the sense sequence.

- A pair of nucleotide sequences consisting of SEQ ID NO: 68 as the antisense sequence and SEQ ID NO: 69 as the sense sequence.

- A pair of nucleotide sequences consisting of SEQ ID NO: 72 as the antisense sequence and SEQ ID NO: 73 as the sense sequence.

- A pair of nucleotide sequences consisting of SEQ ID NO: 76 as the antisense sequence and SEQ ID NO: 77 as the sense sequence.

- A pair of nucleotide sequences consisting of SEQ ID NO: 78 as the antisense sequence and SEQ ID NO: 79 as the sense sequence.

- A pair of nucleotide sequences consisting of SEQ ID NO: 84 as the antisense sequence and SEQ ID NO: 85 as the sense sequence.

- A pair of nucleotide sequences consisting of SEQ ID NO: 86 as the antisense sequence and SEQ ID NO: 87 as the sense sequence.

- A pair of nucleotide sequences consisting of SEQ ID NO: 98 as the antisense sequence and SEQ ID NO: 99 as the sense sequence.

- A pair of nucleotide sequences consisting of SEQ ID NO: 116 as the antisense sequence and SEQ ID NO: 117 as the sense sequence.

[0023] More preferably, the miRNA of the present embodiment comprises one of the following base pairs.

- A pair of nucleotide sequences consisting of SEQ ID NO: 52 as the antisense sequence and SEQ ID NO: 53 as the sense sequence.

- A pair of nucleotide sequences consisting of SEQ ID NO: 72 as the antisense sequence and SEQ ID NO: 73 as the sense sequence.

- A pair of nucleotide sequences consisting of SEQ ID NO: 76 as the antisense sequence and SEQ ID NO: 77 as the sense sequence.

- A pair of nucleotide sequences consisting of SEQ ID NO: 78 as the antisense sequence and SEQ ID NO: 79 as the sense sequence.

[0024] In addition, the miRNA sequence of the present embodiment may comprise a nucleotide sequence selected from the group consisting of SEQ ID NOs: 29-51 shown in Table 2.

Table 2. MicroRNA sequences designed for the human PLP1 gene

SEQID NO: pre-miRNA Sequence 29 AGAAACACAATCCAGTGGCCAGTTTTGGCCACTGACTGACTGGCCACTATTGTGTTTCT thirty AAATAGGTCTCAATTAGCTTTGTTTTGGCCACTGACTGACAAAGCTAAGAGACCTATTT 31 AAGAAACACAATCCAGTGGCCGTTTTGGCCACTGACTGACGGCCACTGTTGTGTTTCTT 32 TAAACAGGTGGAAGGTCATTTGTTTTGGCCACTGACTGACAAATGACCCCACCTGTTTA 33 TTGTAGTCGCCAAAGATCTGCGTTTTGGCCACTGACTGACGCAGATCTGGCGACTACAA 34 AATTAGAGCCTCCATTCCTTTGTTTTGGCCACTGACTGACAAAGGAATAGGCTCTAATT 35 TTAAGGACGGCAAAGTTGTAAGTTTTGGCCACTGACTGACTTACAACTGCCGTCCTTAA 36 TTTAAGGACGGCAAAGTTGTAGTTTTGGCCACTGACTGACTACAACTTCCGTCCTTAAA 37 ATGTCTTTGGGACTCTGACTCGTTTTGGCCACTGACTGACGAGTCAGACCCAAAGACAT 38 TATCTATCCTGTGTCTACCAGGTTTTGGCCACTGACTGACCTGGTAGACAGGATAGATA 39 AAATTACTTTCTGATTCCTCAGGTTTTGGCCACTGACTGACCTGAGGATGAAAGTAATTT 40 TCTAACAAGCCCATGTCTTTGGTTTTGGCCACTGACTGACCAAAGACAGGCTTGTTAGA 41 AATTACTTTCTGATTCCTCAGGGTTTTGGCCACTGACTGACCCTGAGGAAGAAAGTAATT 42 AGTAAATGTACACAGGCACAGGTTTTGGCCACTGACTGACCTGTGCCTGTACATTTACT 43 TAAGTAAGGTTGGCTGAGTTAGTTTTGGCCACTGACTGACTAACTCAGAACCTTACTTA 44 TTCTGTGGGTGAAAGATCCTTGTTTTGGCCACTGACTGACAAGGATCTCACCCACAGAA 45 AGAAGATGCTGACAACACCCTGTTTTGGCCACTGACTGACAGGGTGTTCAGCATCTTCT 46 AATTGTAGCCGGCTGGCTAGTGTTTTGGCCACTGACTGACACTAGCCACGGCTACAATT 47 AGATTTGGGCAAACGCTCTTAGTTTTGGCCACTGACTGACTAAGAGCGTGCCCAAATCT 48 ATTCTACGCTCCCTTATGCTGGTTTTGGCCACTGACTGACCAGCATAAGAGCGTAGAAT 49 TGGTAATAGAGAGACCAGAATGTTTTGGCCACTGACTGACATTCTGGTCTCTATTACCA 50 TATAGATGGCAAGAGGACCAAGTTTTGGCCACTGACTGACTTGGTCCTTGCCATCTATA 51 AAACCAGTGTAGCTGCAGCCCGTTTTGGCCACTGACTGACGGGCTGCATACACTGGTTT

[0025] The miRNA of the present embodiment may contain a nucleotide sequence selected from the group consisting of SEQ ID NOs: 2-7 and 24-51, preferably it may contain a nucleotide sequence selected from the group consisting of SEQ ID NOs: 2- 5, 7, 24, 26, 29-32, 35, 36, 42 and 51, and more preferably it may contain a nucleotide sequence selected from the group consisting of SEQ ID NOS: 2, 29, 31 and 32.

[0026] [Vector]

The oligodendrocyte-specific vector of the present embodiment contains an oligodendrocyte-specific promoter and/or microRNA specific for the PLP1 gene. The vector of the present embodiment is a vector that can be used to treat a disease associated with an abnormality of the PLP1 gene or suppression of the expression of the PLP1 gene.

[0027] The vector may be a plasmid or a vector capable of infecting human cells and expressing a gene, examples of the vector include adeno-associated virus (AAV), lentivirus, retrovirus, adenovirus, Sendai virus and plasmids (including complexes with liposomes or polymers), preferably the vector is a AAV. AAV is a small, non-helper-enveloped virus that belongs to the Dependovirus genus of the Parvoviridae family and infects animals such as humans, primates, and rodents. AAV induces a very weak immune response, the pathogenicity of this virus has not been confirmed. AAV can infect both dividing and non-dividing cells, and after infection of the host cell, AAV can survive outside the nuclear chromosomes and express the gene at a low frequency of insertion into the host's chromosomal genome.

[0028] The AAVs are preferably, for example, any of the serotypes AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8 and AAV9, AAV hybrid mosaic vectors and AAV chimeric vectors can also be preferably used. For example, any combination of AAV1-AAV9 may be present in an AAV hybrid mosaic vector, including an AAV1/2 hybrid. Examples of chimeric AAV vectors include Olig001 (Powell SK et al., Gene Ther. 2016 Nov; 23 (11): 807-814), which has a high tropism for oligodendrocytes.

[0029] The oligodendrocyte-specific promoter included in the vector of the present embodiment is not limited to a particular promoter, and may be any promoter capable of specifically functioning in oligodendrocytes, and preferably the promoter of the present embodiment, that is, the oligodendrocyte-specific promoter, is is an oligodendrocyte-specific promoter containing a nucleic acid at least 90% identical to the nucleotide sequence described in SEQ ID NO: 1. The microRNA specific for the PLP1 gene included in the vector of the present embodiment is not limited to a specific miRNA and may is any microRNA capable of repressing the expression of the PLP1 gene, and preferably it is a microRNA of the present embodiment, that is, a microRNA containing a nucleotide sequence pair consisting of an antisense sequence and a sense sequence, where a pair of nucleotide sequences is selected from the group consisting of pairs containing the antisense sequence indicated in the left column of table 1, and the sense sequence indicated in the right column of the table next to the antisense sequence.

[0030] The vector of the present embodiment may contain the oligodendrocyte-specific promoter of the present embodiment and a transgene operably linked to the promoter. The transgene is not limited to a specific transgene, but it is preferable to use, for example, a microRNA sequence specific for the PLP1 gene, in particular a microRNA sequence specific for the human PLP1 gene. A microRNA sequence specific for the human PLP1 gene can be designed using software such as BLOCK-iT(TM) RNAi Designer (ThermoFisher Scientific, USA). Alternatively, to construct a miRNA sequence specific for the human PLP1 gene, artificial miRNAs are prepared as described in the Examples section and the optimal sequence is selected from them and used to construct miRNAs. As such a microRNA specific for the human PLP1 gene, for example, the microRNA described above of the present embodiment is preferably used.

[0031] The vector of the present embodiment may contain an oligodendrocyte-specific promoter and a miRNA of the present embodiment operably linked to the promoter. The oligodendrocyte-specific promoter is not limited to a particular promoter, but examples thereof include the myelin basic protein gene promoter and the PLP1 gene promoter. As the oligodendrocyte-specific promoter, the above-described oligodendrocyte-specific promoter of this embodiment is preferably used. The vector according to the present embodiment may preferably contain the oligodendrocyte-specific promoter according to the present embodiment and one or more microRNAs according to the present embodiment operably linked to the promoter.

[0032] The vector of the present embodiment may also contain a reporter gene for confirming gene expression. Examples of a reporter gene include, without limitation, GFP, Venus, and tdTomato.

[0033] The vector of the present embodiment can be easily engineered by genetic engineering. For example, an AAV1/2 hybrid vector containing the hCNP promoter described in SEQ ID NO: 1 and a reporter gene can be obtained by the following method. The hCNP promoter encoding a region of a reporter gene such as Venus, the 3' untranslated region, and the SV40 poly-A signal are placed in that order between the two NotI recognition regions of the self-complementary AAV vector, and then the nucleotide sequence of the miRNA expression cassette is inserted into the 3' untranslated region . The vector can be stored frozen at -20°C until use, and when using the vector, AAV293 cells (Takara Bio Inc., Japan) are transfected with the vector along with the serotype-specific helper plasmid and adenoviral helper plasmid pHelper and cultured for 72 hours to provide amplification in cells or in culture medium. Depending on the scale, purification can be carried out by column chromatography, ultrafiltration, ultracentrifugation, and the like. The vector genome titer of the resulting AAV vector can be determined by quantitative PCR using the AAVpro titration kit (Takara Bio Inc., Japan).

[0034] [Pharmaceutical composition]

The pharmaceutical composition of the present embodiment includes the above-described vector of the present embodiment. The pharmaceutical composition of the present embodiment is preferably a pharmaceutical composition for treating a disease associated with an abnormality of the PLP1 gene. Examples of diseases associated with abnormalities in the PLP1 gene include Pelizeus-Merzbacher disease (PMD) and type 2 spastic paraplegia, both of which are caused by a duplication of the PLP1 gene, a point mutation in the PLP1 gene, a deletion or insertion mutation in the PLP1 gene, or multiple sclerosis. The pharmaceutical composition of the present embodiment may also be a PLP1 gene expression inhibitor capable of suppressing PLP1 gene expression.

[0035] The pharmaceutical composition of the present embodiment is preferably an aqueous solution containing an AAV vector, and may have a titer of 5×10 ¹⁰ to 5×10 ¹⁴ vg/ml, preferably 5×10 ¹¹ to 5×10 ¹³ vg/ ml.

[0036] The pharmaceutical composition of the present embodiment may contain a pharmaceutically acceptable auxiliary component such as phosphate buffered saline (PBS), and in addition, it may contain another therapeutic agent. The pharmaceutical composition of the present embodiment can be prepared by dissolving in an aqueous solution such as water, adding other ingredients as needed.

[0037] In the treatment of Peliceus-Merzbacher disease, the pharmaceutical composition of the present embodiment can be administered directly to the brain, and examples of such administration include administration to one or more areas of the brain parenchyma (e.g., white matter of the cerebral hemisphere, internal capsule, cerebellum) or spinal cord, in addition, you can enter intracerebroventricular/intrathecal or intravascular/intraperitoneal. The introduction can be carried out at any time after birth. The amount of drug administered may vary depending on the site of administration and age, but in the case of administration to the brain parenchyma, 0.1 to 2 ml/site can be administered, preferably about 0.5 ml/site. In the case of intracerebroventricular/intrathecal administration, 0.5 to 2 ml/kg body weight can be administered, preferably up to 1 ml/kg body weight. In the case of intravascular or intraperitoneal administration, 1 to 10 ml/kg of body weight can be administered, preferably up to 5 ml/kg of body weight.

[0038] [Method of treatment]

The method of treatment of the present embodiment is a method of treating a disease associated with an abnormality of the PLP1 gene, comprising administering an effective amount of the vector of the present embodiment described above to a patient in need of treatment. Alternatively, the method of treating the present embodiment is a method of suppressing PLP1 gene expression, comprising administering an effective amount of the vector of the present embodiment described above to a patient in need of treatment.

EXAMPLES

[0039] Further, the present invention is described in more detail in the Examples section. However, the present invention is not limited to the following examples.

[0040] <Example 1 Construction of an oligodendrocyte-specific AAV expression cassette and an AAV vector containing it>

Pre-miRNA sequence (PLP1 miRNA, SEQ ID NO: 9) specific for mouse PLP1 gene mRNA (NM_011123.3, SEQ ID NO: 8) and negative control pre-miRNA sequence (miR-neg, SEQ ID NO: 10) are designed using BLOCK-iT(TM) RNAi Designer (ThermoFisher Scientific, USA). miR-neg is a miRNA sequence that can form a hairpin structure and thus be processed into a mature miRNA, but is not expected to be specific to known vertebrate genes.

PLP1 miRNA (SEQ ID NO: 9):

5'-ACTCCAAAGAAACACAATCCAGTTTTGGCCACTGACTGACTGACTGGATTGTTTCTTTGGAGT-3'

miR-neg (SEQ ID NO: 10):

5'-GTATGCATCGAATGAGATTCCGTTTTGGCCACTGACTGACGGAATCTCTCGATGCATAC-3'

[0041] PLP1 microRNA sequence and miR-neg sequence are synthesized in accordance with the designed sequences and cloned in the cloning site of the pcDNA(TM) 6.2-GW/miR vector (ThermoFisher Scientific, USA), which is part of the kit for obtaining the BLOCK-iT expression vector (TM) Pol II miR RNAi. The 5' flanking sequence (SEQ ID NO: 11) and the 3' flanking sequence (SEQ ID NO: 12) derived from mouse miR-155 are located on either side of the PLP1 miRNA sequence or the miR-neg sequence, respectively. The 5' flanking sequence and the 3' flanking sequence derived from mouse miR-155 are the 5' region and the 3' region of mouse miR-155, except for the hairpin fragment and the pre-miRNA sequence or miR-neg placed between the 5' flanking sequence and the 3' flanking sequence so that said miRNA sequences can be expressed.

5'-flanking sequence (SEQ ID NO: 11):

5'-CTGGAGGCTTGCTGAAGGCTGTATGCT-3'

3-'flanking sequence (SEQ ID NO: 12):

5'-CAGGACACAAGGCCTGTTACTAGCACTCACATGGAACAAATGGCC-3'

[0042] Using the obtained vector pcDNA(TM) 6.2-GW/miR, the fragment of the 5'-flanking sequence-PLP1 microRNA sequence (or miR-neg sequence)-3'-flanking sequence is amplified by PCR, the amplification product is digested with XhoI/BamHI and inserted into a DNA fragment obtained by cleaving the vector pW-CAG-Venus-WPRE (SEQ ID NO: 13) with XhoI/BamHI, cleavage is carried out at both ends of the WPRE located at the 3'-end of the venus sequence (SEQ ID NO: 14 ) to remove the WPRE sequence. The resulting vector is then cleaved with SpeI/EcoRI to remove the CAG promoter, the DNA fragment is inserted into this region at the 5' end of the Venus sequence, where the DNA fragment is obtained by PCR amplification of the 1.8 kb human CNP promoter (SEQ ID NO: 1). o., and are cleaved at both ends of SpeI/EcoRI to allow for oligodendrocyte-specific gene expression. The resulting vector is further cleaved with NotI to remove the backbone of a single-stranded AAV derived from pW-CAG-Venus-WPRE, coupled to a self-complementary adeno-associated virus (scAAV) backbone obtained by cleaving pscW-PABPN1 (SEQ ID NO: 15) with NotI to give final microRNA constructs Pscw-hCNP-Venus-PLP1 (SEQ ID NO: 16) and Pscw-hCNP-Venus-miR-neg (SEQ ID NO: 17) (Fig. 1).

[0043] An AAV1/2 fusion vector (scAAV-AAV1/2 vector) is produced by the method described in Non-Patent Document 1. Namely, AAV293 cells are co-transfected using PEI, previously obtained scAAV constructs, Pscw-hCNP-Venus-PLP1 miRNA or Pscw-hCNP-Venus-miR-neg, serotype-specific AAV helper plasmids p5E18RXC1 (Xiao W et al., J Virol (1999) 73: 3994-4003), and pAAV-RC (Agilent Technologies) encoding the rep and cap genes AAV1 and AAV2, respectively, and the adenoviral helper plasmid pHelper (Agilent Technology). Cells were harvested 72 hours after transfection and the scAAV-AAV1/2 vector was purified using the AAVpro purification kit (Takara Bio Inc., Japan). Titers of vector genomes were determined by quantitative PCR using the AAVpro titration kit (Takara Bio Inc., Japan).

[0044] The resulting scAAV-AAV1/2 vectors were constructed as AAV-hCNP-Venus-PLP1 miRNA vector (or PLP1 microRNA vector) and AAV-hCNP-Venus-miR-neg vector (or miR-neg vector).

[0045] <Example 2. Administration of AAV vectors to wild-type mice and expression of said vectors>

One microliter solution of the PLP1 miRNA vector or miR-neg vector obtained in Example 1 (titer: 1.2×10 ¹² vg/ml) was injected into the striatum and inner capsule of each 10 day old wild-type Jcl:B6C3F1 mouse (n= 5/group). Before injection, mice are anesthetized with pentobarbital solution. The introduction of the vector is carried out using a 33G syringe at a rate of 150 nl/min. After injection, each mouse is left alone for 1 minute and then the needle is slowly withdrawn from the brain. Thereafter, the mice are returned as a group to their mother and reared up to the 17th day after birth.

[0046] The expression of the gene provided by each vector is determined by the detection of green fluorescent protein at day 17 after birth. Venus-expressing cell types are identified by double immunostaining with cell markers such as Gst-π (marker for mature oligodendrocytes), NeuN (marker for neurons), Iba1 (marker for microglia), and GFAP (marker for astrocytes).

[0047] To conduct immunostaining, Jcl:B6C3F1 mice are anesthetized with Foran inhalation solution, after which they are subjected to transcardial perfusion with PBS and then with fresh 4% paraformaldehyde solution. Brains are harvested, fixed overnight at 4° C., then the medium is replaced with 30% sucrose in PBS and stored frozen in OCT Compound (TissueTech, Inc.) enclosing medium. Get coronal sections (20 μm) and subject them to immunostaining to confirm the specificity of infected cells expressing Venus. Sections are incubated with blocking solution (PBS containing 0.05% Triton(TM) X-100 and 5% goat serum) for 3 hours at room temperature, followed by incubation at 4°C overnight with Gst-π (Cell Signaling Technology, Inc., 1:300), GFAP (Millipore Corporation, 1:400), NeuN (Chemicon, 1:400) or Iba1 (Biocare Medical, LLC., 1:500) as primary antibodies. The sections are then washed and incubated with the appropriate secondary antibody (ThermoFisher Scientific, USA) for 1 hour at room temperature. Cell nuclei are visualized with 40,60-diamidino-2-phenylindole (DAPI, Sigma-Aldrich Co. LLC). Slides are embedded in ProLong® Diamond histological medium (Thermo Fisher Scientific, USA) and photographed using a KEYENCE fluorescence microscope (KEYENCE CORPORATION, Japan).

[0048] The results are shown in FIG. 2. From FIG. 2 shows that Venus-positive cells correspond to Gst-π-positive oligodendrocytes, but do not correspond to NeuN-positive neurons, GFAP-positive astrocytes, and IbaI-positive microglia. Thus, FIG. 2 demonstrates that the miR-neg vector is expressed in an oligodendrocyte-specific manner with high efficiency.

[0049] <Example 3. Evaluation 1 of the effectiveness of suppression of expression (knockdown) in terms of the level of expression of the PLP1 gene - Part 1>

One microliter solution of each of the two scAAV-AAV1/2 vectors (titer: 1.2×10 ¹² vg/ml) prepared in Example 1 was injected into the striatum, internal capsule and cerebellum of each 10 day old Jcl:B6C3F1 wild-type mouse (n=3/group). Use the same method of introduction as in example 2. Then, to evaluate the effectiveness of knockdown miRNA PLP1, determine the expression of PLP1 protein in Venus-positive cells by immunostaining. Immunostaining was carried out in the same manner as in Example 2, except that a rabbit anti-PLP1 polyclonal antibody was used as the primary antibody (Numata Y et al., J Biol Chem. 2013 Mar 15; 288 (11): 7451-66 ), and a fluorescent antibody against rabbit immunoglobulins (Thermo Fisher Scientific, USA) is used as a secondary antibody. Each area of the corpus callosum, striatum, and internal capsule is photographed using a KEYENCE fluorescence microscope (KEYENCE CORPORATION, Japan).

[0050] The mean fluorescence intensity of immunostained PLP1 was quantified using Image J 1.45s (Wayne Rasband, National Institutes of Health, USA), the results of the quantitative analysis of fluorescence intensity by PLP1 immunostaining are shown in FIG. 3. From FIG. 3, it can be seen that there is almost no difference in PLP1 protein expression between the side not injected with the miR-neg vector (uninfected side) and the side injected with the vector (infected side), while in the case of the PLP1 microRNA vector, the expression inhibition effect PLP1 protein observed on the infected side compared to the uninfected side is 40%. The absence of a difference in the number of astrocytes and microglia between the uninfected side and the infected side indicates the absence of a reactive inflammatory response when AAV is administered.

[0051] <Example 4. Evaluation of the effectiveness of suppression of expression (knockdown) in terms of the level of expression of the PLP1 gene - Part 2>

One microliter solution of each of the two scAAV-AAV1/2 vectors (titer: 1.2×10 ¹² vg/ml) prepared in Example 1 was injected into the striatum and internal capsule of each 10 day old Jcl:B6C3F1 wild-type mouse (n =4/group). Use the same method of introduction as in example 2. Then, to evaluate the efficiency of PLP1 miRNA knockdown, determine the expression of PLP1 mRNA in Venus-positive cells by quantitative PCR.

[0052] In order to sort Venus-positive cells, Jcl:B6C3F1 mice are anesthetized with Foran inhalation solution and subjected to transcardial perfusion with 20 ml of PBS. The brain is removed and roughly cut with microscissors. 1 ml of Accutase (Millipore Corporation) was added to each brain and incubated at 37° C. for 30 minutes. 2 ml of Hank's balanced salt solution (HBSS) containing 10% FBS is added to each sample and the tissue is homogenized with a micropipette. Each sample is filtered through a 100 µm cell sieve and the collected cell suspension is centrifuged. To clear the cells of myelin residues, they are resuspended in HBSS containing 40% Percoll (Amersham plc) and centrifuged at 700 g for 25 minutes at room temperature. The top layer of myelin is aspirated and the monocytes are resuspended in 1 ml of DMEM/F12 medium. Cells were separated on a FACSCanto flow cytometer (BD Biosciences) and the results analyzed using FlowJo software (Tree Star Inc., OR, RRID: NIF-0000 to 30575). The results are presented as a percentage of the total number of cells.

[0053] For quantitative PCR, total RNA is isolated from Venus-positive cells using the RNeasy Mini kit (Qiagen), and for cDNA synthesis, the SuperScript® reverse transcriptase kit (Thermo Fisher Scientific, USA) is used. Gene expression was analyzed by quantitative RT-PCR with a LightCycler® 480 SYBR Green I Master and a LightCycler® 480 Instrument using the following specific primers (F. Hoffmann-La Roche Ltd., Switzerland). The results are normalized to the β-actin housekeeping gene and the relative expression levels of PLP1 and Olig2 are calculated. The CP values (crosspoint values) obtained for each sample are converted to logarithmic values to plot relative expression levels±SD, and the resulting plots are shown in FIG. four.

PLP1 forward primer (SEQ ID NO: 18): 5'-GTTCCAGAGGCCAACATCAAGCTC-3'

Reverse primer PLP1 (SEQ ID NO: 19): 5'-AGCCATACAACAGTCAGGGCATAG-3'

Forward primer Olig2 (SEQ ID NO: 20): 5'-GGGAGGTCATGCCTTACGCGC-3'

Reverse primer Olig2 (SEQ ID NO: 21): 5'-CTCCAGCGAGTTGGTGAGC-3'

β-actin forward primer (SEQ ID NO: 22): 5'-CACAGCTTCTTTGCAGCTCCTT-3'

β-actin reverse primer (SEQ ID NO: 23): 5'-GACGACCAGCGCAGCGATA-3'

[0054] From FIG. 4, it can be seen that there are no significant differences in the expression level of the mRNA of the Olig2 oligodendrocyte marker between the miR-neg vector and the miRNA PLP1 (B) vector, while the level of PLP1 mRNA expression after the introduction of the PLP1 miRNA vector indicates the presence of an effect of suppressing the expression of the PLP1 gene, which is approximately 60% (A).

[0055] <Example 5: Effect of administration to PLP1 transgenic mice (PLP1-Tg)>

PLP1 miRNA vector (treatment group, n=16) or miR-neg vector (treatment sham group , n=16) to test the treatment. PLP1-Tg/B6C3 is obtained by backcrossing PLP1-Tg/BDF1 donated by Dr. Tetsushi Kagawa of the National Institute of Physiological Sciences with the B6C3 strain (Kagawa T et al., Neuron. 1994 Aug; 13 (2): 427-42). In the case of PLP1-Tg homozygous individuals, 1 µl of a solution of each of the two kinds of scAAV-AAV1/2 vectors (titer: 1.2×10 ¹² vg/ml) obtained in Example 1 was injected into the striatum and inner capsule of each mouse of age 10 days in the same manner as in example 2. Wild-type littermates (n=16) injected with the miR-neg vector were used as positive controls. Some of these mice have their brain tissue removed at day 25 post-natal (15 days post-injection) and histological analysis (immunostaining and morphometric analysis) of the brain tissue is performed, the rest of the mice are recorded for weight and survival.

[0056] Histological analysis by immunostaining is carried out as follows. In wild-type mice, PLP1-Tg mice from the treatment group and PLP1-Tg mice from the sham treatment group, where n=5 in each group, each of the corpus callosum, striatum and internal capsule were immunostained according to the method described in example 3, 25 days after birth (15 days after injection). In wild-type mice, which constitute the positive control group, after injection of PLP1, fibrous strongly stained images are observed corresponding to the myelin sheaths of the corpus callosum, striatum and internal capsule. In the analysis of PLP1-Tg mice, stained images corresponding to myelin sheaths are virtually absent in mice receiving sham treatment, although a stained image was obtained indicating abnormal accumulation of PLP1 in the cytoplasm of oligodendrocytes. On the other hand, mice in the treatment group had almost no staining images indicative of PLP1 accumulation in the cytoplasm, but fibrosis staining was observed, which corresponded to the myelin sheath, thus it was found that the introduction of the PLP1 microRNA vector gave improved staining images of PLP1 compared to abnormal. In FIG. 5 shows micrographs of the corpus callosum.

[0057] Micromorphological analysis using an electron microscope is carried out as follows. In WT mice and PLP1-Tg mice from the treatment group and the sham treatment group, where n=2/group, fixation with reflux was performed using 0.1 M phosphate buffer containing 2% glutaraldehyde and 2% paraformaldehyde on day 25 after birth (day 15 post-injection), and then the brain is harvested and further fixed by soaking in the same buffer. Each of the corpus callosum, striatum, and internal capsule is examined for AAV-infected areas under a stereoscopic fluorescent microscope, a 600 µm square piece of tissue is cut out and fixed in 0.1 M phosphate buffer containing 1% osmium. After dehydration, the tissue section is immersed in epoxy resin, an ultrathin section of 70 nm is obtained, which is observed using an electron microscope (Tecnai Spirit, Thermo Fisher Scientific, USA) at a magnification of 7300 times.

[0058] FIG. 6 shows photographs taken with an electron microscope. It appears that in the corpus callosum, striatum, and internal capsule of wild-type mice, most axons are myelinated. In the PLP1-Tg mice from the sham treatment group, the proportion of myelinated axons was clearly low, in addition, the axons were constricted. On the other hand, in the PLP1-Tg mice of the treatment group, the proportion of myelinated axons increased, and the axon diameter significantly exceeded that of the mice in the sham treatment group. From the obtained results, it can be seen that the analysis of ultrafine morphology indicates that the introduction of the PLP1 miRNA vector has a therapeutic effect due to an increase in the proportion of myelinated fibers.

[0059] Mice from the treatment group and mice from the sham treatment group, where n=9/group, are observed from 25 days of age to death, assessing body weight and survival in each group. The administration of PLP1-Tg to mice was found to produce a significant survival (lifespan) effect (FIG. 7) and an increase in body weight (FIG. 8), confirming the effectiveness of this treatment method.

[0060] The described results demonstrate that an oligodendrocyte-specific promoter and an AAV vector containing this promoter can be used as a fundamental method for developing a method for treating PMD, and that an AAV gene expression vector containing PLP1-specific miRNA and this promoter, is an effective treatment for PMD.

[0061] <Example 6: Evaluation of the effectiveness of suppression of expression (knockdown) in terms of the level of expression of the PLP1 gene - Part 3>

To evaluate the knockdown efficiency of human PLP1 miRNA constructed using BLOCK-iT(TM) RNAi Designer (Thermo Fisher Scientific, USA), the pcDNA(TM) 6.2-hPLP1 plasmid, which can overexpress the human PLP1 coding sequence, and the plasmid pscw.CAG.Venus.PLP1-miRNA obtained by cloning the sequence in the 3'-untranslated region downstream of the cDNA encoding Venus, a fluorescent protein expressed under the control of the CAG promoter, was simultaneously introduced into HeLa cells by transfection. TransIt-LT1 (Takara Bio Inc.) was used for transfection. After 24 hours, cells are harvested and total RNA is extracted with the RNeasy Mini Kit (Qiagen). For quantitative PCR, cDNA is synthesized using the SuperScript(R) reverse transcriptase kit (Thermo Fisher Scientific, USA). Gene expression was analyzed by quantitative PCR with a LightCycler (R) 480 SYBR Green I Master and LightCycler (R) 480 Instrument using the following specific primers (F. Hoffmann-La Roche Ltd., Switzerland). The primers used to determine the expression of the PLP1 gene by quantitative PCR are described below.

Human PLP1 forward primer (SEQ ID NO: 280): 5'-GCTCCAACCTTCTGTCCATCT-3'

Human PLP1 reverse primer (SEQ ID NO: 281): 5'-ACGGCAAAGTTGTAAGTGGC-3'

Human β-actin forward primer (SEQ ID NO: 282): 5'-GACAGGATGCAGAAGGAGATTACT-3'

Human β-actin reverse primer (SEQ ID NO: 283): 5'-TGATCCACATCTGCTGGAAGGT-3'

[0062] In FIG. 9 shows the knockdown results. Knockdown efficiency is calculated by taking the expression level of PLP1 in miR-neg expressing cells as 1. Results are normalized to the β-actin housekeeping gene and calculated as relative expression level±SD. Seven sequences are identified that reduce the expression level of the PLP1 gene (SEQ ID NOs: 2, 3, 4, 5, 7, 24 and 26).

[0063] <Example 7. Evaluation of the effectiveness of suppression of expression (knockdown) in terms of the level of expression of the PLP1 gene - Part 4>

An siRNA pair consisting of the sense strand and the corresponding antisense strand shown in Table 3 and the transfection solution (RNAi MAX, Invitrogen) are mixed, the resulting solution is added to the U-251MG cells, after which the cells are cultured for 48 hours.

[Table 3]

Table 3. Designed siRNA sequences for the human PLP1 gene

No. SEQID NO: Sense/antisense strand miRNA sequence 1 118 hPLP1-001 UGACCUUCCACCCUGUUUAUdTdT 119 hPLP1-001anti AUAAACAGGUGGAAGGUCAdTdT 2 120 hPLP1-002 GUGCCUGUGUACAUUUACUdTdT 121 hPLP1-002anti AGUAAAUGUACACAGGCACdTdT 3 122 hPLP1-003 UUGCCCAAAUCUGCCUAUUdTdT 123 hPLP1-003anti AAUAGGCAGAUUUUGGGCAAdTdT four 124 hPLP1-004 AGGAAUGGAGGCUCUAAUUdTdT 125 hPLP1-004anti AAUUAGAGCCUCCAUUCCUdTdT five 126 hPLP1-005 UGGCCACUGGAUUGUGUUUdTdT 127 hPLP1-005anti AAACACAAUCCAGUGGCCAdTdT 6 128 hPLP1-006 GCGGGUGUGUCAUUGUUUGdTdT 129 hPLP1-006anti CAAACAAUGACACACCGCGCdTdT 7 130 hPLP1-007 GACCUUCCACCUGUUUAUUdTdT 131 hPLP1-007anti AAUAAACAGGUGGAAGGUCdTdT 8 132 hPLP1-008 ACUCCCUUCUCCUUGAUAAdTdT 133 hPLP1-008anti UUAUCAAGGAGAAGGGAGUdTdT nine 134 hPLP1-009 CUGCAAGUCACAAAGGAAUdTdT 135 hPLP1-009anti AUUCCUUUGUGACUUGCAGdTdT 10 136 hPLP1-010 GGUCCUCUUGCCAUCUAUAdTdT 137 hPLP1-010anti UAUAGAUGGCAAGAGGACCdTdT eleven 138 hPLP1-011 CCUCGUUAGGGAAGAGAAAdTdT 139 hPLP1-011anti UUUCUCUUCCCUAACGAGGdTdT 12 140 hPLP1-012 GGAGAAGAGGACAAAGAUAdTdT 141 hPLP1-012anti UAUCUUUGUCCCUCUUCUCCdTdT 13 142 hPLP1-013 GGCCAACAUCAAGCUCAUUdTdT 143 hPLP1-013anti AAUGAGCUUGAUGUUGGCCdTdT fourteen 144 hPLP1-014 AGGCCAACAUCAAGCUCAUdTdT 145 hPLP1-014anti AUGAGCUUGAUGUUGGCCUdTdT 15 146 hPLP1-015 CUCCCUUCUCCUUGAUAACdTdT 147 hPLP1-015anti GUUAUCAAGGAGAAGGGAGdTdT sixteen 148 hPLP1-016 GCCUCUUUCUUCUUCCUUUdTdT 149 hPLP1-016anti AAAGGAAGAAGAAAAGAGGCdTdT 17 150 hPLP1-017 CUGUGCCUGUGUACAUUUAdTdT 151 hPLP1-017anti UAAAUGUACACAGGCACAGdTdT eighteen 152 hPLP1-018 AAGGAAUGGAGGCUCUAAUdTdT 153 hPLP1-018anti AUUAGAGCCUCCAUUCCUUdTdT nineteen 154 hPLP1-019 AGGGUGUUGUCAGCAUCUUdTdT 155 hPLP1-019anti AAGAUGCUGACAACACCCCUdTdT 20 156 hPLP1-020 ACAGCUGAGUUCCAAAUGAdTdT 157 hPLP1-020anti UCAUUUGGAACUCAGCUGUdTdT

No. SEQID NO: Sense/antisense strand miRNA sequence 21 158 hPLP1-021 GCCACUGGAUUGUGUUUCUdTdT 159 hPLP1-021anti AGAAACACAAUCCAGUGGCdTdT 22 160 hPLP1-022 ACAACUUUGCCGUCCUUAAdTdT 161 hPLP1-022anti UUAAGGACGGCAAAGUUGUdTdT 23 162 hPLP1-023 GCUGAUGCCAGAAUGUAUGdTdT 163 hPLP1-023anti CAUACAUUCUGGCAUCAGCdTdT 24 164 hPLP1-024 UUGCCGUCCUUAAAACUCAUdTdT 165 hPLP1-024anti AUGAGUUUAAGGACGGCAAdTdT 25 166 hPLP1-025 CUGCCUCUUUCUUCUUCCUdTdT 167 hPLP1-025anti AGGAAGAAAGAAAGAGGCAGdTdT 26 168 hPLP1-026 CCACUGGAUUGUGUUUCUUdTdT 169 hPLP1-026anti AAGAAACACAAUCCAGUGGdTdT 27 170 hPLP1-027 GGAUCAGAAAGUAAUUUCUdTdT 171 hPLP1-027anti AGAAAUUACUUUCUGAUCCdTdT 28 172 hPLP1-028 UAGCCAGCCGGCUACAAUUdTdT 173 hPLP1-028anti AAUUGUAGCCGGCUGGCUAdTdT 29 174 hPLP1-029 AGCGUAGAAUCUGUGUAGAdTdT 175 hPLP1-029anti UCUACACAGAUUCUACGCUdTdT thirty 176 hPLP1-030 UCCCUUCUCCUUGAUAACAdTdT 177 hPLP1-030anti UGUUAUCAAGGAGAAGGGAdTdT 31 178 hPLP1-031 UUCCAAGGAGCUGAGAAUAdTdT 179 hPLP1-031anti UAUUCUCAGCUCCUUGGAAdTdT 32 180 hPLP1-032 UGCCAGAAUGUAUGGUGUUdTdT 181 hPLP1-032anti AACACCAUACAUUCUGGCAdTdT 33 182 hPLP1-033 AUGCCUUCCAGUAUGUCAUdTdT 183 hPLP1-033anti AUGACAUACUGGAAGGCAUdTdT 34 184 hPLP1-034 CAACUUUGCCGUCCUUAAAdTdT 185 hPLP1-034anti UUUAAGGACGGCAAAGUUGdTdT 35 186 hPLP1-035 CCAACAUCAAGCUCAUUCUdTdT 187 hPLP1-035anti AGAAUGAGCUUGAUGUUGGdTdT 36 188 hPLP1-036 UGGCCUUACACCUCGUUAGdTdT 189 hPLP1-036anti CUAACGAGGUGUAAGGCCAdTdT 37 190 hPLP1-037 AGGACAUCCCGACAAGUUUdTdT 191 hPLP1-037anti AAACUUGUCGGGAUGUCCCUdTdT 38 192 hPLP1-038 GUCAGAGUCCCAAAGACAUdTdT 193 hPLP1-038anti AUGUCUUUGGGACUCUGACdTdT 39 194 hPLP1-039 UUGCAGGAGAAGAGGACAAdTdT 195 hPLP1-039anti UUGUCCUCUUCUCCUGCAAdTdT 40 196 hPLP1-040 GGUAGACACAGGAUAGAUAdTdT 197 hPLP1-040anti UAUCUAUCCUGUGUCUACCdTdT

No. SEQID NO: Sense/antisense strand miRNA sequence 41 198 hPLP1-041 UCAGCCAACCUUACUUACAdTdT 199 hPLP1-041anti UGUAAGUAAAGGUUGGCUGAdTdT 42 200 hPLP1-042 AUGGAACUGCCUCUUUCUUdTdT 201 hPLP1-042anti AAGAAAGAGGCAGUUCCAUdTdT 43 202 hPLP1-043 ACUCAGCCAACCUUACUUAdTdT 203 hPLP1-043anti UAAGUAAGGUUGGCUGAGUdTdT 44 204 hPLP1-044 CUUCCACUGAUGGAAAACAAdTdT 205 hPLP1-044anti UUGUUUCCAUCAGUGGAAGdTdT 45 206 hPLP1-045 AUGACCUUCCACCCUGUUUAdTdT 207 hPLP1-045anti UAAACAGGUGGAAGGUCAUdTdT 46 208 hPLP1-046 UCUCCUGAGGAUCAGAAAGdTdT 209 hPLP1-046anti CUUUCUGAUCCUAGGAGAdTdT 47 210 hPLP1-047 GAGGAUCAGAAAGUAAUUUdTdT 211 hPLP1-047anti AAAUUACUUUCUGAUCCUCdTdT 48 212 hPLP1-048 GGAUCUUUCACCCACAGAAdTdT 213 hPLP1-048anti UUCUGUGGGUGAAAGAUCCdTdT 49 214 hPLP1-049 CUGAGGAUCAGAAAGUAAUdTdT 215 hPLP1-049anti AUUACUUUCUGAUCCUAGdTdT 50 216 hPLP1-050 CUGGUAGACACAGGAUAGAdTdT 217 hPLP1-050anti UCUAUCCUGUGUCUACCAGdTdT 51 218 hPLP1-051 GCAGUUGCUGGUGGCUAAUdTdT 219 hPLP1-051anti AUUAGCCACCAGCAACUGCdTdT 52 220 hPLP1-052 AAGACAUGGGCUUGUUAGAdTdT 221 hPLP1-052anti UCUAACAAGCCCAUGUCUUdTdT 53 222 hPLP1-053 UUGCUGCCACUUACAACUUdTdT 223 hPLP1-053anti AAGUUGUAAGUGGCAGCAAdTdT 54 224 hPLP1-054 UCGUUAGGGAAGAGAAACAdTdT 225 hPLP1-054anti UGUUUCUCUUCCCUAACGAdTdT 55 226 hPLP1-055 UCCACUGAUGGAAAACAAAGdTdT 227 hPLP1-055anti CUUUGUUUCCAUCAGUGGAdTdT 56 228 hPLP1-056 AGAGCGUUUGCCCAAAUCUdTdT 229 hPLP1-056anti AGAUUUGGGCAAACGCUCUdTdT 57 230 hPLP1-057 UGAUUGCUGCCACUUACAAdTdT 231 hPLP1-057anti UUGUAAGUGGCAGCAAUCAdTdT 58 232 hPLP1-058 GGAGCGUAGAAUCUGUGUADTdT 233 hPLP1-058anti UACACAGAUUCUACGCUCCdTdT 59 234 hPLP1-059 UGAGGAUCAGAAAGUAAUUdTdT 235 hPLP1-059anti AAUUACUUUCUGAUCCUCAdTdT 60 236 hPLP1-060 GGUGUUGUCAGCAUCUUCUdTdT 237 hPLP1-060anti AGAAGAUGCUGACAACACCdTdT No. SEQID NO: Sense/antisense strand miRNA sequence 61 238 hPLP1-061 GUCAAAGCAAGGAUCUUUCdTdT 239 hPLP1-061anti GAAAGAUCCUUGCUUUGACdTdT 62 240 hPLP1-062 AGAUCUUUGGCGACUACAAdTdT 241 hPLP1-062anti UUGUAGUCCGCCAAAGAUCUdTdT 63 242 hPLP1-063 UGCAGGAGAAGAGGACAAAdTdT 243 hPLP1-063anti UUUGUCCUCUUCUCCUGCAdTdT 64 244 hPLP1-064 UGGUGGCUAAUGGUGUAACdTdT 245 hPLP1-064anti GUUACACCAUUAGCCACCAdTdT 65 246 hPLP1-065 AGCUAAUUGAGACCUAUUUdTdT 247 hPLP1-065anti AAAUAGGUCUCAAUUAGCUdTdT 66 248 hPLP1-066 GUGCUGAUGCCAGAAUGUADTdT 249 hPLP1-066anti UACAUUCUGGCAUCAGCACdTdT 67 250 hPLP1-067 UUUCCAAGGAGCUGAGAAUdTdT 251 hPLP1-067anti AUUCUCAGCUCCUUGGAAAdTdT 68 252 hPLP1-068 UGUGCCUGUGUACAUUUACdTdT 253 hPLP1-068anti GUAAAUGUACACAGGCACAdTdT 69 254 hPLP1-069 CAGGUACAGGAAGGAAUGdTdT 255 hPLP1-069anti CAUUCCUUCUCUGUACCUGdTdT 70 256 hPLP1-070 GCAUAAGGGAGCGUAGAAUdTdT 257 hPLP1-070anti AUUCUACGCUCCCUUAUGCdTdT 71 258 hPLP1-071 UCAGGUACAGGAAGGAAUdTdT 259 hPLP1-071anti AUUCCUUCUCUGUACCUGAdTdT 72 260 hPLP1-072 GCUGCAGCUACACUGGUUUdTdT 261 hPLP1-072anti AAACCAGUGUAGCUGCAGCdTdT 73 262 hPLP1-073 UCUCCCAUGGAAUGCUUUCdTdT 263 hPLP1-073anti GAAAGCAUUCCAUGGGAGAdTdT 74 264 hPLP1-074 UGCCUUCCAGUAUGUCAUCdTdT 265 hPLP1-074anti GAUGACAUACUGGAAGGCAdTdT 75 266 hPLP1-075 GCUGCCACUUACAACUUUGdTdT 267 hPLP1-075anti CAAAGUUGUAAGUGGCAGCdTdT 76 268 hPLP1-076 ACUUUGCCGUCCUUAAACUdTdT 269 hPLP1-076anti AGUUUAAGGACGGCAAAGUdTdT 77 270 hPLP1-077 ACCUUACUUACAGCAUAAGdTdT 271 hPLP1-077anti CUUAUGCUGUAAGUAAGGUdTdT 78 272 hPLP1-078 UGGACUACUGAAGCCCUAAdTdT 273 hPLP1-078anti UUAGGGCUUCAGUAGUCCAdTdT 79 274 hPLP1-079 CUUCCAGUAUGUCAUCUAUdTdT 275 hPLP1-079anti AUAGAUGACAUACUGGAAGdTdT 80 276 hPLP1-080 UCUGGUCUCUCUAUUACCAdTdT 277 hPLP1-080anti UGGUAAUAGAGAGACCAGAdTdT 81 278 hPLP1-081 GUCCCAAAGACAUGGGCUUdTdT 279 hPLP1-081anti AAGCCCAUGUCUUUGGGACdTdT

[0064] RNA is then extracted from the cells and PLP1 gene expression is assessed by quantitative PCR (n=3). SuperPrep Cell Lysis RT qPCR Kit (TOYOBO CO., LTD.) is used for RNA preparation and cDNA synthesis. For quantitative PCR, a Taqman probe for the PLP1 gene (Hs00166914_m1, Applied Biosystems) and the S18 gene (Hs99999901_s1, Applied Biosystems) was used. Gene expression results are normalized to the housekeeping gene S18.

[0065] FIG. 10 shows the results. On the vertical axis of Fig. 10 defer the knockdown efficiency for U-251MG cells to which only the transfection solution was added. PLP1 gene knockdown efficiency is calculated as relative expression levels, taking the expression level in U-251MG cells to which only the transfection solution is added as 1. Twenty-four pairs of siRNA sequences are selected that reduce the expression level of the PLP1 gene to a mean value of 50% or less in both trials (No. 2, 4, 10, 16, 21, 22, 26, 28, 34, 38). 40, 43, 45, 47, 48, 52, 56, 59, 60, 62, 65, 70, 72 and 80).

[0066] <Example 8 Evaluation of the effectiveness of suppression of expression (knockdown) in terms of the level of expression of the PLP1 gene - Part 5

The miRNA sequence was designed based on the 24 miRNA sequence pairs obtained in Example 7 (Table 2). Among them, miRNA sequences specific to the sequence encoding the human PLP1 gene sequence are selected (SEQ ID NOs: 2, 29, 30, 31, 32, 33, 35, 36, 42 and 51). Among them, SEQ ID NOs: 2, 29, 31, 32, and 33 are sequence-specific sequences for common mice and for primates such as the common marmoset.

[0067] To evaluate 10 miRNA sequences, each plasmid obtained by cloning any of the sequences downstream of the CAG promoter is transfected into HeLa cells that overexpress the human PLP1 coding sequences. The experimental details of the primers used to analyze PLP1 gene expression by qPCR are the same as in Example 6. The knockdown result is shown in FIG. 11. On the vertical axis of FIG. 11 plots the expression level±SD, relative to the expression level when the vector containing miR-neg is added, where the PLP1 expression level is taken as 1. Referring to FIG. 11, microRNAs having any of SEQ ID NOs: 2, 29, 30, 31, 32, 35, 36, 42, and 51 knock out the human PLP1 gene.

[0068] <Example 9. Evaluation of the effectiveness of suppression of expression (knockdown) in terms of the level of expression of the PLP1 gene - Part 6>

Each of the vectors used in Examples 6 and 8 containing miRNA having the nucleotide sequence described in one of SEQ ID NOs: 2, 29, 31, 32 and 35 are transfected into U-251MG cells that endogenously express the human PLP1 gene, and evaluate the efficiency of suppressing the expression of the PLP1 gene. The plasmid obtained by cloning downstream of the CAG promoter is introduced into cells by transfection, and only cells expressing GFP are selected by FACS. Thereafter, PLP1 gene expression was analyzed by quantitative PCR. As primers, the nucleotide sequences described in SEQ ID NO: 280-283 are used.

[0069] The results are shown in FIG. 12. On the vertical axis of FIG. 12 plot relative expression levels, taking PLP1 expression level as 1 when a vector containing miR-neg is added. According to FIG. 12, vectors containing miRNAs of SEQ ID NOs: 2, 29, 31, 32, and 35 also have an inhibitory effect on the expression of the endogenous PLP1 gene.

[0070] <Example 10 Evaluation of the effectiveness of suppression of expression (knockdown) in terms of the level of expression of the PLP1 gene - Part 7>

One microliter of the scAAV-AAV1/2 vector solution containing the miRNA of SEQ ID NO: 2 prepared in Example 9 (titer: 1.2×10 ¹² vg/ml) was injected into the striatum, internal capsule, and cerebellum of each of the Jcl mice :B6C3F1 wt, 10 days old (n=3/group). The same administration method as in Example 2 was used. After that, in order to evaluate the efficiency of PLP1 miRNA knockdown, PLP1 protein expression in Venus-positive cells was determined by immunostaining. The immunostaining method involves using a rabbit anti-PLP1 polyclonal antibody (Numata Y et al., J Biol Chem. 2013 Mar 15; 288 (11): 7451-66) as a primary antibody, and a fluorescent anti-rabbit immunoglobulin antibody as a secondary antibody ( Thermo Fisher Scientific, USA). Each area of the corpus callosum, striatum, and internal capsule is photographed using a KEYENCE fluorescent microscope (KEYENCE CORPORATION, Japan).

[0071] The mean fluorescence intensity of immunostained PLP1 is quantified using J 1.45s image (Wayne Rasband, National Institutes of Health, USA) and FIG. 13 shows the results of quantitative analysis of fluorescence intensity by immunostaining. On the vertical axis of Fig. 13 plot the relative levels of PLP1 expression on the infected side compared to the uninfected side. From FIG. 13, it can be seen that there is almost no difference in PLP1 protein expression between the side not injected with the miR-neg vector (uninfected side) and the side injected with the vector (infected side), while in the case of the PLP1 microRNA vector, an inhibitory effect is observed expression of the PLP1 protein on the infected side, which is 40% compared to the uninfected side.

Application in industry

[0072] The promoter of the present invention and the AAV vector of the present invention can be used as therapeutic agents against PMD caused by PLP1 duplication. They can also be used as therapeutic agents for PMD caused by PLP1 point mutation. In addition, since PLP1 is one of the target antigens in multiple sclerosis, it is possible that they can be used as therapeutic agents against multiple sclerosis.

Claims (21)

1. MicroRNA specific for the PLP1 gene, containing a pair of nucleotide sequences consisting of an antisense sequence and a sense sequence, where the pair of nucleotide sequences is selected from the group consisting of pairs of antisense sequences shown in the left column of the table below and sense sequences shown next to antisense sequences in the right column of the table below: Antisense sequence semantic sequence AAAGGAAGAAGAAAAGAGGCAG (SEQ ID NO: 52) CTGCCTCTCTTCTTCCTTT (SEQ ID NO: 53) AACACCAGGAGCCACACAACG (SEQ ID NO: 54) CGTTGTGTCTCCTGGTGTT (SEQ ID NO: 55) TTCCATGGGAGAACACCATAC (SEQ ID NO: 56) GTATGGTGCTCCCATGGAA (SEQ ID NO: 57) TGAGCAGGGAAACCAGTGTAG (SEQ ID NO: 58) CTACACTGTTCCCTGCTCA (SEQ ID NO: 59) ACCCCAAAGAAACACAATCCA (SEQ ID NO: 62) TGGATTGTTTCTTTGGGGT (SEQ ID NO: 63) ACAAATGCAGCAATAAACAGG (SEQ ID NO: 64) CCTGTTTAGCTGCATTTGT (SEQ ID NO: 65) AAAGAATGAGCTTGATGTTGG (SEQ ID NO: 68) CCAACATCGCTCATTCTTT (SEQ ID NO: 69) AGAAACACAATCCAGTGGCCA (SEQ ID NO: 72) TGGCCACTATTGTGTTTCT (SEQ ID NO: 73) AAATAGGTCTCAATTAGCTTT (SEQ ID NO: 74) AAAGCTAAGAGACCTATTT (SEQ ID NO: 75) AAGAAACACAATCCAGTGGCC (SEQ ID NO: 76) GGCCACTGTTGTGTTTCTT (SEQ ID NO: 77) TAAACAGGTGGAAGGTCATTT (SEQ ID NO: 78) AAATGACCCCACCTGTTTA (SEQ ID NO: 79) TTAAGGACGGCAAAGTTGTAA (SEQ ID NO: 84) TTACAACTGCCGTCCTTAA (SEQ ID NO: 85) TTTAAGGACGGCAAAGTTGTA (SEQ ID NO: 86) TACAACTTCCGTCCTTAAA (SEQ ID NO: 87) AGTAAATGTACACAGGCACAG (SEQ ID NO: 98) CTGTGCCTGTACATTTACT (SEQ ID NO: 99) AAACCAGTGTAGCTGCAGCCC (SEQ ID NO: 116) GGGCTGCATACACTGGTTT (SEQ ID NO: 117) 2. MicroRNA according to claim 1, containing a nucleotide sequence selected from the group consisting of SEQ ID NOs: 2-5, 7, 24, 26, 29-32, 35, 36, 42 and 51. 3. MicroRNA according to claim 1, where the pair of nucleotide sequences is selected from the group consisting of pairs of the antisense sequence indicated in the left column of the table below and the sense sequence indicated to the right of the antisense sequence in the right column of the table below: Antisense sequence semantic sequence AAAGGAAGAAGAAAAGAGGCAG (SEQ ID NO: 52) CTGCCTCTCTTCTTCCTTT (SEQ ID NO: 53) AGAAACACAATCCAGTGGCCA (SEQ ID NO: 72) TGGCCACTATTGTGTTTCT (SEQ ID NO: 73) AAGAAACACAATCCAGTGGCC (SEQ ID NO: 76) GGCCACTGTTGTGTTTCTT (SEQ ID NO: 77) TAAACAGGTGGAAGGTCATTT (SEQ ID NO: 78) AAATGACCCCACCTGTTTA (SEQ ID NO: 79) 4. MicroRNA according to claim 1, containing a nucleotide sequence selected from the group consisting of SEQ ID NOs: 2, 29, 31 and 32. 5. A vector for suppressing the expression of the PLP1 gene, containing the miRNA sequence according to any one of paragraphs. 1-4, in which the miRNA sequence is operably linked to an oligodendrocyte-specific promoter. 6. The vector according to claim 5, wherein the oligodendrocyte-specific promoter contains a nucleic acid at least 90% identical to the nucleotide sequence described in SEQ ID NO: 1. 7. The vector according to claim 6, wherein the oligodendrocyte-specific promoter contains a nucleic acid having the nucleotide sequence described in SEQ ID NO: 1. 8. Vector according to any one of paragraphs. 5-7, which is an adeno-associated virus (AAV) vector. 9. Pharmaceutical composition for the treatment of a disease associated with an abnormality of the PLP1 gene, containing the vector according to any one of paragraphs. 5-8 and a pharmaceutically acceptable additional component. 10. The pharmaceutical composition according to claim 9, wherein the disease is Pelizeus-Merzbacher disease, type 2 spastic paraplegia, or multiple sclerosis. 11. The pharmaceutical composition according to claim 9, wherein the disease is Peliceus-Merzbacher disease. 12. An inhibitor of the expression of the PLP1 gene, suppressing the expression of the PLP1 gene, containing the vector according to any one of paragraphs. 5-8. 13. A method of treating a disease associated with an abnormality of the PLP1 gene, comprising the introduction of an effective amount of the vector according to any one of paragraphs. 5-8 to a patient in need of treatment. 14. The method of claim 13, wherein the disease is Pelizeus-Merzbacher disease, type 2 spastic paraplegia, or multiple sclerosis. 15. The method of claim 13, wherein the disease is Pelizeus-Merzbacher disease. 16. A method of suppressing the expression of the PLP1 gene, including the introduction of an effective amount of the vector according to any one of paragraphs. 5-8 to a patient in need of treatment. 17. Vector according to any one of paragraphs. 5-8 for use in a method of treating a disease associated with an abnormal PLP1 gene. 18. The vector of claim 17, wherein the disease is Pelizeus-Merzbacher disease, type 2 spastic paraplegia, or multiple sclerosis. 19. The vector of claim 17, wherein the disease is Pelizeus-Merzbacher disease.

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