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Oligodendrocyte-specific promoter, mirna specific to plp1 gene, vector including said promoter and/or mirna, and pharmaceutical composition including said vector

A technology of oligodendrocytes and PLP1, which is applied in the field of PLP1 gene-specific miRNA, can solve problems such as no records, and achieve the effect of inhibiting expression

Pending Publication Date: 2020-09-22
NAT CENT OF NEUROLOGY & PSYCHIATRY +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] Non-Patent Documents 1 and 2 disclose gene therapy using AAV vectors and the promoters used therein, but there is no description of gene therapy for PLP1 gene abnormalities and the promoters or miRNAs used therein

Method used

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  • Oligodendrocyte-specific promoter, mirna specific to plp1 gene, vector including said promoter and/or mirna, and pharmaceutical composition including said vector
  • Oligodendrocyte-specific promoter, mirna specific to plp1 gene, vector including said promoter and/or mirna, and pharmaceutical composition including said vector
  • Oligodendrocyte-specific promoter, mirna specific to plp1 gene, vector including said promoter and/or mirna, and pharmaceutical composition including said vector

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0134]

[0135] BLOCK-iT TM RNAi Designer (ThermoFisher Scientific, USA) was used to design a precursor miRNA sequence (PLP1 miRNA, sequence number 9) targeting the mRNA of the mouse PLP1 gene (NM_011123.3, sequence number 8) and as a negative control The precursor miRNA sequence (miR-neg, SEQ ID NO: 10). It should be noted that miR-neg is a miRNA sequence that is presumed to form a hairpin structure and be processed into a mature miRNA, but does not target a known vertebrate gene.

[0136] PLP1-miRNA (SEQ ID NO: 9):

[0137]5'-ACTCCAAAAGAAACACAATCCAGTTTTGGCCACTGACTGACTGGATTGTTTCTTTGGAGT-3'

[0138] miR-neg (sequence number 10):

[0139] 5'-GTATGCATCGAATGAGATTCCGTTTTGGCCACTGACTGACGGAATCTCTCGATGCATAC-3'

[0140] The PLP1 miRNA sequence and miR-neg sequence were synthesized according to the design, and cloned into the cloning site of the pcDNA (trademark) 6.2-GW / miR vector (ThermoFisherScientific, USA) contained in the BLOCK-iT (trademark) Pol IImiR RNAi expression vector k...

Embodiment 2A

[0148]

[0149] To the unilateral striatum and internal capsule of wild-type mouse Jcl on the 10th day after birth: B6C3F1, respectively inject 1 μl of the solution of the PLP1 miRNA carrier or miR-neg carrier obtained in Example 1 (titer: 1.2 ×10 12 vg / ml) (n=5 for each group). Before injection, mice were anesthetized with pentobarbital solution. Injection of the vector was performed at a rate of 150 nl / min using a 33G syringe. After injection, the needle was left in place for 1 minute, and the needle was slowly withdrawn from the brain. Afterwards, they were sent back to the mother mice in a group to raise them until the 17th day after birth.

[0150] Gene expression from the vector was assessed by detecting the green fluorescent protein Venus at postnatal day 17. Venus-expressing cells were identified by double immunostaining with cellular markers including Gst-π (mature oligodendrocytes), NeuN (neurons), Iba1 (microglia), and GFAP (astrocytes) kind of.

[0151] For...

Embodiment 3

[0153]

[0154] To the wild-type mouse Jcl on the 10th day after birth: B6C3F1 unilateral striatum, internal capsule and cerebellum respectively inject 1 μl of the solution of the two scAAV-AAV1 / 2 vectors obtained in Example 1 (titer : 1.2×10 12 vg / ml) (n=3 for each group). The injection method is the same as in Example 2. Thereafter, in order to evaluate the PLP1 miRNA knockdown efficiency, the expression of PLP1 protein in Venus-positive cells was investigated by immunostaining. Regarding the immunostaining method, an anti-PLP1 rabbit polyclonal antibody (Numata Y et al., J Biol Chem. 2013 Mar 15; 288(11): 7451-66) was used as the primary antibody, and an anti-rabbit fluorescent antibody (ThermoFisher Scientific, USA) was used. Except for the second antibody, it was carried out in the same manner as in Example 1. Nuclei of the corpus callosum, striatum, and internal capsule were photographed using a KEYENCE fluorescence microscope (KEYENCE, Japan).

[0155] The average...

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Abstract

The purpose of the present invention is to provide a vector, a promoter and miRNA for the same, and a pharmaceutical composition including the vector, whereby expression of the PLP1 gene can be suppressed specifically in oligodendrocytes in order to treat PMD caused by an abnormality in the PLP1 gene. This oligodendrocyte-specific promoter includes a nucleic acid having at least 90% sequence identity with the base sequence represented by SEQ ID NO: 1. This miRNA specific to the PLP1 gene has a pair of base sequences comprising a predetermined antisense sequence and sense sequence.

Description

technical field [0001] The present invention relates to an oligodendrocyte-specific promoter, particularly an oligodendrocyte-specific promoter comprising a nucleic acid having at least 90% sequence identity with the nucleotide sequence described in SEQ ID NO: 1. The present invention additionally relates to PLP1 gene-specific miRNAs. The present invention further relates to a vector comprising the above-mentioned promoter and / or the above-mentioned miRNA, and a pharmaceutical composition comprising the vector. Background technique [0002] Congenital leukodysplasia is a general term for children's brain diseases that occur due to poor development of white matter in the brain. Currently, 11 diseases are known. A representative disease among them is Pelizaeus-Merzbacher disease (PMD). PMD is characterized by a myelination disorder of the central nervous system. It is a rare intractable disease that causes severe motor developmental disorders and neurological symptoms soon a...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/113A61K31/7088A61K35/761A61K48/00C12N15/861
CPCC12N15/113A61K31/7088A61P25/14C12N15/86A61K48/0058C12N2750/14143C12N2830/008C12Y301/04037C12N2310/14Y02A50/30A61K35/761A61K48/00A61P25/00A61P21/00C12N2310/141C12N2320/32
Inventor 井上健李珩冈田尚巳大木优小泉诚
Owner NAT CENT OF NEUROLOGY & PSYCHIATRY
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