JP2017197443A - Composition for treating tdp-43 proteinopathy - Google Patents
Composition for treating tdp-43 proteinopathy Download PDFInfo
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- JP2017197443A JP2017197443A JP2016087320A JP2016087320A JP2017197443A JP 2017197443 A JP2017197443 A JP 2017197443A JP 2016087320 A JP2016087320 A JP 2016087320A JP 2016087320 A JP2016087320 A JP 2016087320A JP 2017197443 A JP2017197443 A JP 2017197443A
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Abstract
Description
本発明はTDP-43プロテイノパチー治療用組成物に関する。 The present invention relates to a composition for treating TDP-43 proteinopathy.
筋萎縮性側索硬化症（amyotrophic lateral sclerosis：以下、本明細書ではしばしば「ALS」と表記する）は、孤発性及び遺伝性（家族性）が知られる難治性の神経変性疾患である。大脳、脳幹、脊髄の運動ニューロンの病変によって上位運動ニューロン障害、下位運動ニューロン障害が進行し、それに伴う筋萎縮と筋力低下の原因によって上肢の機能障害、歩行障害、構音障害、嚥下障害、呼吸障害等の症状が現われる。若年及び中年で多く発症するが、治療法は限定的な対処療法に過ぎず、その効果も低い。例えば、人工呼吸器により比較的長期間生存する例も知られてはいるが、病勢の進展が比較的速いため、通常は発症後平均3.5年で呼吸筋の麻痺等によって死亡する。 Amyotrophic lateral sclerosis (hereinafter often referred to as “ALS” in the present specification) is an intractable neurodegenerative disease known to be sporadic and hereditary (familial). Upper motor dysfunction and lower motoneuron disorders progress due to cerebral, brain stem and spinal motor neuron lesions, and upper limb dysfunction, gait disorder, articulation disorder, dysphagia, respiratory disorder Symptoms such as appear. It develops more often in young and middle-aged, but treatment is only limited treatment and is less effective. For example, although it is known that the ventilator can survive for a relatively long period of time, the disease progresses relatively quickly, and usually die on the average of 3.5 years after the onset due to paralysis of the respiratory muscles.
一方、前頭側頭葉変性症（frontotemporal lobar degeneration：以下、本明細書ではしばしば「FTLD」と表記する）は、脳前頭葉及び側頭葉の神経変性を伴う認知症である。64歳未満で発症する若年性認知症の中では、血管性認知症及びアルツハイマー病に次いで多く、早期から行動障害、脱抑制的徴候、感情障害、言語障害等の症状が現われる。FTLDは、ALSと同様に孤発性及び遺伝性が知られる他、ALS症例では5〜10％がFTLD症状をきたし、またFTLD症例では10〜30％がALS類似の運動神経症状を呈することも報告されている。さらに、ALSとユビキチン陽性封入体を伴う前頭側頭葉変性症（FTLD-U）は、共に神経細胞内にユビキチン陽性封入体を形成する。このように、ALSとFTLDは、臨床的にも、病理学的にも、そして遺伝学的にも多くの共通性が見られる（非特許文献３）。 On the other hand, frontotemporal lobar degeneration (hereinafter often referred to as “FTLD” in the present specification) is a dementia associated with neurodegeneration of the frontal and temporal lobes. Among juvenile dementia that develops before the age of 64 years, it is the second most common after vascular dementia and Alzheimer's disease, and symptoms such as behavioral disorder, disinhibitory sign, emotional disorder, and language disorder appear from an early stage. FTLD is known to be sporadic and heritable, similar to ALS, and 5-10% of ALS cases have FTLD symptoms, and 10-30% of FTLD cases may have motor neuron symptoms similar to ALS. It has been reported. Furthermore, frontotemporal lobar degeneration (FTLD-U) with ALS and ubiquitin-positive inclusions together form ubiquitin-positive inclusions in neurons. Thus, ALS and FTLD have many commonalities clinically, pathologically, and genetically (Non-patent Document 3).
2006年に新井ら及びNeumannらによりALSとFTLD-Uにおけるユビキチン陽性封入体の共通構成成分としてTAR DNA結合タンパク質43（TAR DNA-binding protein of 43kDa：以下、本明細書ではしばしば「TDP-43」と表記する）が同定された（非特許文献１及び２）。
As a common component of ubiquitin-positive inclusion bodies in ALS and FTLD-U by Arai et al. And Neumann et al. In 2006, TAR DNA-binding protein 43 (TAR DNA-binding protein of 43kDa: hereinafter referred to as "TDP-43" (Referred to as
TDP-43は、広範に発現するDNA/RNA結合タンパク質であって、正常な細胞では核内のRNA転写において、pre-mRNAスプライシング及びマイクロRNA（miRNA）プロセシングを制御している。さらに、近年、TDP-43が核内外をシャトルして細胞質内及び神経軸索でもmRNAの輸送等に関与し、それによって運動ニューロンの生存維持を制御することが推測されている（非特許文献４）。しかし、その機能の詳細については未だに解明されていない。 TDP-43 is a widely expressed DNA / RNA-binding protein that controls pre-mRNA splicing and microRNA (miRNA) processing in nuclear RNA transcription in normal cells. Furthermore, in recent years, it has been speculated that TDP-43 shuttles inside and outside the nucleus and participates in mRNA transport in the cytoplasm and nerve axons, thereby controlling the survival maintenance of motor neurons (Non-Patent Document 4). ). However, the details of its function have not yet been elucidated.
TDP-43は、大部分のALSやFTLDにおいて、核内には認められず、神経細胞の細胞質内に異常沈着している。しかし、核内TDP-43の消失やTDP-43の細胞質内異常蓄積及びそれに伴う局在変化がALSやFTLDの発症原因とどのように関連するのか、その具体的な機序については明らかにされていない。 TDP-43 is not found in the nucleus in most ALS and FTLD, but is abnormally deposited in the cytoplasm of nerve cells. However, the specific mechanism of the disappearance of TDP-43 in the nucleus, abnormal accumulation of TDP-43 in the cytoplasm, and associated localization changes with the onset of ALS and FTLD has been clarified. Not.
上記のように、ALSやFTLDは、発症原因や病態進行機序に不明な点が多く、それ故に効果的な治療薬が未だに存在しない。したがって、両疾患の治療や改善に有効性の高い新薬の開発が強く求められている。 As described above, ALS and FTLD have many unclear points regarding the cause of onset and the pathological mechanism, and therefore, there are no effective therapeutic agents yet. Therefore, there is a strong demand for the development of new drugs that are highly effective in treating and improving both diseases.
TDP-43の異常により発症するALS及びFTLD等は、TDP-43プロテイノパチー（proteinopathy：タンパク質症）と呼ばれる。本発明の課題は、TDP-43プロテイノパチーの治療薬を開発し、提供することである。 ALS and FTLD that develop due to abnormalities in TDP-43 are called TDP-43 proteinopathy. An object of the present invention is to develop and provide a therapeutic agent for TDP-43 proteinopathy.
神経細胞は、軸索や樹状突起等の長い神経突起を有するが、その生存、構造若しくは機能維持、神経回路形成、神経再生時におけるシナプス形成、及び突起伸長のためには、合成されたタンパク質を神経突起内で即座に供給しなければならない。そのためには、それらのタンパク質をコードする遺伝子が核内で発現した後、そのmRNAを神経突起内にまで輸送し、局所翻訳をする必要がある。したがって、標的mRNAの神経突起内輸送は、極めて重要である。 Nerve cells have long neurites such as axons and dendrites, but are synthesized proteins for their survival, structure or function maintenance, neural circuit formation, synapse formation during nerve regeneration, and process extension. Must be supplied immediately within the neurite. To that end, after the genes encoding these proteins are expressed in the nucleus, it is necessary to transport the mRNA into the neurite and perform local translation. Therefore, intraneurite transport of target mRNA is extremely important.
一方、TDP-43プロテイノパチーの神経細胞では、細胞質におけるTDP-43の異常沈着や局在変化によって神経細胞質内の遊離TDP-43量が相対的に低下する。 On the other hand, in TDP-43 proteinopathy neurons, the amount of free TDP-43 in the neurocytoplasm is relatively decreased due to abnormal deposition and localization of TDP-43 in the cytoplasm.
本発明者らは、神経細胞における遊離TDP-43量の相対的低下によって、必要なmRNAの神経突起内輸送が障害され、神経突起へのmRNAの供給が不足する結果、神経変性を生じる、というTDP-43プロテイノパチー発症機序の仮説を構築した。もしも、TDP-43により神経突起内輸送が制御される標的遺伝子が明らかになれば、TDP-43プロテイノパチーの有効な治療薬となり得る。実際、TDP-43が神経突起内に存在することやneurofilament L等のmRNAを輸送することは既に報告されている（Volkening K., et al., 2009, Brain Res, 1305:168）。しかし、TDP-43により神経突起内へ輸送される標的mRNAを網羅的に検索した報告は、知られていない。 The inventors have stated that the relative decrease in the amount of free TDP-43 in neurons impairs the required intraneurite transport of mRNA, resulting in a lack of mRNA supply to neurites resulting in neurodegeneration. We developed a hypothesis for the mechanism of TDP-43 proteinopathy. If a target gene whose intraneurite transport is regulated by TDP-43 is clarified, it can be an effective therapeutic agent for TDP-43 proteinopathy. In fact, it has already been reported that TDP-43 exists in neurites and transports mRNA such as neurofilament L (Volkening K., et al., 2009, Brain Res, 1305: 168). However, there is no known report that exhaustively searches for target mRNAs transported into neurites by TDP-43.
そこで、本発明者らは、上記仮説を検証するために神経細胞内のTDP-43発現量を抑制して神経突起へのmRNA供給障害を生じたTDP-43プロテイノパチー疑似神経細胞を作製し、その神経突起内における各種mRNA量を網羅的に検証した。その結果、疑似神経細胞では、正常神経細胞と比較して多数のリボソームタンパク質mRNAの量が有意に低減していた。この結果は、TDP-43の輸送標的遺伝子がリボソームタンパク質遺伝子であることを示唆している。実際、TDP-43プロテイノパチーではTDP-43によるmRNAの輸送障害により神経突起の伸長障害を生じることや、リボソームタンパク質遺伝子の過剰発現によりTDP-43の発現低下による神経突起の伸長障害を阻止できることも明らかになった。本願発明は、これらの研究成果に基づくものであって、以下を提供する。 Therefore, in order to verify the above hypothesis, the present inventors produced a TDP-43 proteinopathy pseudo-neuron that produced an impaired mRNA supply to the neurite by suppressing the expression level of TDP-43 in the neuron, Various mRNA levels in neurites were comprehensively verified. As a result, the amount of many ribosomal protein mRNAs was significantly reduced in the pseudo-neurons compared to the normal neurons. This result suggests that the transport target gene of TDP-43 is a ribosomal protein gene. In fact, it is clear that TDP-43 proteinopathy can prevent neurite outgrowth due to impaired TDP-43 mRNA transport, and that ribosomal protein gene overexpression can prevent neurite outgrowth due to decreased TDP-43 expression. Became. The present invention is based on these research results and provides the following.
（１）一以上のリボソームタンパク質若しくはその活性断片又はそれらをコードする核酸を有効成分とするTDP-43プロテイノパチー治療用組成物。
（２）前記リボソームタンパク質が配列番号３〜４８で示すいずれかのアミノ酸配列からなる、（１）に記載のTDP-43プロテイノパチー治療用組成物。
（３）前記リボソームタンパク質をコードする核酸が配列番号４９〜９４で示すいずれかの塩基配列からなるリボソームタンパク質遺伝子である、（１）に記載のTDP-43プロテイノパチー治療用組成物。
（４）前記核酸が遺伝子発現ベクターに含まれている、（１）〜（３）のいずれかに記載のTDP-43プロテイノパチー治療用組成物。
（５）前記TDP-43プロテイノパチーが神経突起伸長障害を伴う疾患である、（１）〜（４）のいずれかに記載のTDP-43プロテイノパチー治療用組成物。
（６）前記TDP-43プロテイノパチーが筋萎縮性側索硬化症又は前頭側頭葉変性症である、（５）に記載のTDP-43プロテイノパチー治療用組成物。
(1) A composition for treating TDP-43 proteinopathy, comprising as an active ingredient one or more ribosomal proteins or active fragments thereof or a nucleic acid encoding them.
(2) The composition for treating TDP-43 proteinopathy according to (1), wherein the ribosomal protein comprises any one of the amino acid sequences represented by SEQ ID NOs: 3 to 48.
(3) The composition for treating TDP-43 proteinopathy according to (1), wherein the nucleic acid encoding the ribosomal protein is a ribosomal protein gene comprising any one of the nucleotide sequences represented by SEQ ID NOs: 49 to 94.
(4) The composition for treating TDP-43 proteinopathy according to any one of (1) to (3), wherein the nucleic acid is contained in a gene expression vector.
(5) The composition for treating TDP-43 proteinopathy according to any one of (1) to (4), wherein the TDP-43 proteinopathy is a disease associated with neurite outgrowth disorder.
(6) The composition for treating TDP-43 proteinopathy according to (5), wherein the TDP-43 proteinopathy is amyotrophic lateral sclerosis or frontotemporal lobar degeneration.
本発明のTDP-43プロテイノパチー治療用組成物によれば、TDP-43プロテイノパチーの治療又は症状改善が期待できる。 According to the composition for treating TDP-43 proteinopathy of the present invention, treatment or symptom improvement of TDP-43 proteinopathy can be expected.
１．概要
本発明は、TDP-43プロテイノパチー治療用組成物（本明細書では、しばしば単に「組成物」と略称する）に関する。本発明の組成物は、リボソームタンパク質を有効成分とする。本発明の組成物によれば、従来対処療法しか知られていなかったALSやFTLD等のTDP-43プロテイノパチーを治療することができる。
1. SUMMARY The present invention relates to a composition for the treatment of TDP-43 proteinopathy (often simply abbreviated as “composition” herein). The composition of the present invention contains a ribosomal protein as an active ingredient. According to the composition of the present invention, it is possible to treat TDP-43 proteinopathy such as ALS and FTLD, which have conventionally been known only for coping therapy.
２．定義
本明細書において頻用する用語について、以下で定義する。
「TAR DNA結合タンパク質-43（TDP-43:TAR DNA-binding Protein 43kDa）」とは、ヘテロ核内リボ核酸タンパク質（heterogeneous nuclear ribonucleoprotein：hnRNP）の一種である。分子内にRNA結合配列であるRRM(RNA Recognition Motif)ドメインを2つ有する。
2. Definitions Terms used frequently in this specification are defined below.
“TAR DNA-binding protein-43 (TDP-43: TAR DNA-binding
本明細書においてTDP-43は、配列番号１で示されるアミノ酸配列からなるヒト野生型TDP-43の他、配列番号１で示されるアミノ酸配列において、1個又は複数個のアミノ酸が付加、欠失、及び／又は置換したアミノ酸配列からなるヒト変異型TDP-43、及び配列番号１で示されるアミノ酸配列、好ましくは2つのRRM以外のアミノ酸配列と85％以上、90％以上、95％以上、97％以上、又は98％以上のアミノ酸同一性を有するアミノ酸配列からなり、かつヒト野生型TDP-43と同等の機能を有するヒト変異型TDP-43又は他種TDP-43オルソログを包含する。 In this specification, TDP-43 is a human wild-type TDP-43 consisting of the amino acid sequence shown in SEQ ID NO: 1, and one or more amino acids are added or deleted in the amino acid sequence shown in SEQ ID NO: 1. And / or human mutant TDP-43 comprising a substituted amino acid sequence, and the amino acid sequence represented by SEQ ID NO: 1, preferably two amino acid sequences other than RRM and 85% or more, 90% or more, 95% or more, 97 It includes human mutant TDP-43 or other species TDP-43 orthologs having an amino acid sequence having% or more or 98% or more amino acid identity and having a function equivalent to that of human wild-type TDP-43.
本明細書において「複数個」とは、2〜10の整数、例えば、2〜7、2〜5、2〜4、2〜3の整数をいう。 In the present specification, the term “plurality” refers to an integer of 2 to 10, for example, an integer of 2 to 7, 2 to 5, 2 to 4, and 2 to 3.
本明細書において「アミノ酸同一性」とは、2つのアミノ酸配列間において、必要に応じて一方又は双方にギャップを導入しながら両配列をアミノ酸の一致数が最大となるようにアラインメントしたときに、一方のアミノ酸配列のアラインメント対象領域における全アミノ酸数に対する他方のアミノ酸配列の前記一致したアミノ酸数の割合（％）をいう。 As used herein, `` amino acid identity '' means that when two amino acid sequences are aligned so that the number of amino acid matches is maximized while introducing gaps in one or both as necessary, The ratio (%) of the number of amino acids corresponding to the other amino acid sequence to the total number of amino acids in the alignment target region of one amino acid sequence.
「TDP-43プロテイノパチー（TDP-43 proteinopathy）」とは、TDP-43遺伝子、その遺伝子発現量、TDP-43 mRNA又はタンパク質の細胞内存在量、又はそれらの局在性の異常を伴う疾患の総称をいう。健常体の細胞内においてTDP-43は、通常、1細胞あたりの遺伝子発現量、TDP-43 mRNA又はタンパク質存在量及びそれらの局在場所が厳格に制御されている（Ayala, Y.M., et al., 2011, EMBO J. 30(2):277-288.）。したがって、それらが通常範囲を逸脱した場合、その個体は何らかの変調をきたし得る。ただし、本明細書におけるTDP-43プロテイノパチーは、TDP-43における前記異常と疾患の関連性が未解明であってもよく、さらにTDP-43における前記異常が、疾患の原因となっているか否かは問わない。 “TDP-43 proteinopathy” is a general term for diseases associated with abnormal TDP-43 gene, its gene expression level, TDP-43 mRNA or protein abundance, or their localization. Say. In healthy cells, TDP-43 is usually tightly controlled in terms of gene expression per cell, TDP-43 mRNA or protein abundance and their location (Ayala, YM, et al. , 2011, EMBO J. 30 (2): 277-288.). Thus, if they deviate from the normal range, the individual can undergo some modulation. However, the TDP-43 proteinopathy in the present specification may not yet elucidate the relationship between the abnormality in TDP-43 and the disease, and whether the abnormality in TDP-43 causes the disease or not. Does not matter.
TDP-43プロテイノパチーには神経変性疾患が挙げられる。例えば、ALS、FTLD（特にFTLD-U）、アルツハイマー病（AD：Alzheimer disease）、又はパーキンソン病（PD：Parkinson disease）が挙げられる。特に神経突起伸長障害及び神経変性を伴うTDP-43プロテイノパチーは本発明の組成物の対象疾患として好適である。例えばALS及びFTLDが挙げられる。 TDP-43 proteinopathy includes neurodegenerative diseases. Examples include ALS, FTLD (particularly FTLD-U), Alzheimer's disease (AD), or Parkinson's disease (PD). In particular, TDP-43 proteinopathy associated with neurite outgrowth disorder and neurodegeneration is suitable as a target disease of the composition of the present invention. Examples include ALS and FTLD.
本明細書において「治療」とは、疾患の治癒、それに伴う症状の緩和又は除去、及び／又は進行の阻止又は抑制をいう。 As used herein, “treatment” refers to the cure of a disease, the alleviation or elimination of symptoms associated therewith, and / or the prevention or suppression of progression.
３．構成
３−1．構成成分
（１）有効成分
本発明の組成物は、一以上のリボソームタンパク質若しくはその活性断片又はそれらをコードする核酸を必須の有効成分として包含する。それぞれの有効成分について以下で具体的に説明をする。
3. Configuration 3-1. Component (1) Active Ingredient The composition of the present invention includes one or more ribosomal proteins or active fragments thereof or nucleic acids encoding them as essential active ingredients. Each active ingredient will be specifically described below.
（リボソームタンパク質及びその活性断片）
「リボソーム」は、ヒトの場合、4種のリボソームRNA（rRNA）及び80種のリボソームタンパク質で構成され、大サブユニット（large subunit）及び小サブユニット（small subunit）の2つのサブユニットからなる二量体複合体である。細胞質内に存在し、mRNAの遺伝情報に基づいた翻訳反応が行われるタンパク質合成の場として機能する。神経細胞においては、神経突起内にも存在し、輸送されてきたmRNAを神経突起内で翻訳し、目的のタンパク質をその場で供給する上で寄与している。
(Ribosomal proteins and active fragments thereof)
In humans, “ribosomes” are composed of 4 types of ribosomal RNA (rRNA) and 80 types of ribosomal proteins, and consists of two subunits, a large subunit and a small subunit. It is a monomer complex. It exists in the cytoplasm and functions as a place for protein synthesis where translation reactions are performed based on mRNA genetic information. In nerve cells, it also exists in neurites, and it contributes to the translation of the transported mRNA in neurites and the supply of the target protein on the spot.
「リボソームタンパク質」は、前述のリボソームを構成するタンパク質である。ヒトにおいては、大サブユニット及び小サブユニットのそれぞれを構成するリボソームタンパク質が計80種知られているが、本発明の組成物の有効成分となり得るリボソームタンパク質は、そのいずれであってもよい。例えば、表１において配列番号３〜４８で示すいずれかのアミノ酸配列からなるリボソームタンパク質が挙げられる。 The “ribosome protein” is a protein that constitutes the ribosome described above. In humans, a total of 80 ribosomal proteins constituting each of the large subunit and the small subunit are known, and any of the ribosomal proteins that can be the active ingredient of the composition of the present invention may be used. For example, a ribosomal protein consisting of any one of the amino acid sequences shown in SEQ ID NOs: 3 to 48 in Table 1 can be mentioned.
表１において、No.1〜27は大サブユニットを構成するリボソームタンパク質であり、またNo.28〜46は小サブユニットを構成するリボソームタンパク質である。 In Table 1, Nos. 1 to 27 are ribosomal proteins constituting the large subunit, and Nos. 28 to 46 are ribosomal proteins constituting the small subunit.
リボソームタンパク質は、その固有の機能を保持する限り、表１において配列番号３〜４８で示すアミノ酸配列に、1又は複数個のアミノ酸が付加、欠失、又は置換したアミノ酸配列、又は前述のアミノ酸配列に対して80％以上又は85％以上、好ましくは90％以上、95％以上、より好ましくは96％以上、97％以上、98％以上又は99％以上のアミノ酸同一性を有するアミノ酸配列からなる変異リボソームタンパク質であってもよい。 As long as the ribosomal protein retains its unique function, one or a plurality of amino acids are added, deleted, or substituted to the amino acid sequence shown in SEQ ID NOs: 3 to 48 in Table 1, or the amino acid sequence described above A mutation comprising an amino acid sequence having 80% or more or 85% or more, preferably 90% or more, 95% or more, more preferably 96% or more, 97% or more, 98% or more, or 99% or more It may be a ribosomal protein.
「（その）活性断片」とは、リボソームタンパク質の一部からなる機能断片で、各リボソームタンパク質に特有の機能を保持し、それ単体で全長リボソームタンパク質と同等以上の活性を示すタンパク質断片をいう。活性断片は、それぞれのリボソームタンパク質の活性を保持する限り、そのアミノ酸長は問わない。 The “(active) fragment” is a functional fragment composed of a part of a ribosomal protein, which retains a function specific to each ribosomal protein, and shows a protein fragment that exhibits an activity equal to or higher than that of a full-length ribosomal protein. As long as the active fragment retains the activity of each ribosomal protein, its amino acid length is not limited.
本明細書においては、リボソームタンパク質及びその活性断片をまとめてしばしば「リボソームタンパク質等」と表記する。 In the present specification, ribosomal proteins and active fragments thereof are often collectively referred to as “ribosomal proteins and the like”.
（それらをコードする核酸）
「それらをコードする核酸」とは、前記リボソームタンパク質又はその活性断片をコードする核酸をいう。本明細書では、しばしば「リボソームタンパク質遺伝子等」と表記する。
(Nucleic acid encoding them)
“Nucleic acid encoding them” refers to a nucleic acid encoding the ribosomal protein or an active fragment thereof. In this specification, it is often written as “ribosome protein gene or the like”.
本明細書において「リボソームタンパク質をコードする核酸」とは、すなわちリボソームタンパク質遺伝子、又はその転写産物であるリボソームタンパク質mRNA及びそれを鋳型として作製されたcDNAをいう。リボソームタンパク質mRNAは、mRNA前駆体（pre-mRNA）及び成熟mRNAのいずれも包含するが、実質的には成熟mRNAを意味する。 As used herein, “nucleic acid encoding a ribosomal protein” refers to a ribosomal protein gene, or a ribosomal protein mRNA that is a transcription product thereof, and a cDNA prepared using it as a template. Ribosomal protein mRNA includes both mRNA precursor (pre-mRNA) and mature mRNA, but substantially refers to mature mRNA.
リボソームタンパク質遺伝子（リボソームタンパク質cDNA）の具体例として、表１において配列番号４９〜９４で示すいずれかの塩基配列からなる遺伝子が挙げられる。また、リボソームタンパク質mRNAには、配列番号４９〜９４で示すいずれかの塩基配列において、t（チミン）をu（ウラシル）に置換した塩基配列からなるmRNAが挙げられる。 Specific examples of the ribosomal protein gene (ribosomal protein cDNA) include genes consisting of any one of base sequences shown in SEQ ID NOs: 49 to 94 in Table 1. Examples of the ribosomal protein mRNA include mRNA having a base sequence in which t (thymine) is substituted with u (uracil) in any one of the base sequences represented by SEQ ID NOs: 49 to 94.
本明細書において「リボソームタンパク質の活性断片をコードする核酸」とは、前述の活性断片をコードする核酸である。DNA又はRNAは問わない。 In the present specification, the “nucleic acid encoding an active fragment of a ribosomal protein” is a nucleic acid encoding the aforementioned active fragment. DNA or RNA is not important.
本発明の組成物においてリボソームタンパク質遺伝子等において、DNAで構成される核酸は、遺伝子発現ベクターに含まれていることが好ましい。 In the ribosomal protein gene or the like in the composition of the present invention, the nucleic acid composed of DNA is preferably contained in a gene expression vector.
「遺伝子発現ベクター」とは、遺伝子や遺伝子断片を発現可能な状態で含み、その遺伝子等の発現を制御できる発現単位をいう。本明細書において「発現可能な状態」とは、プロモーターの制御下である下流域にリボソームタンパク質遺伝子等を配置していることをいう。したがって、プロモーターが活性することでリボソームタンパク質遺伝子等の発現が誘導される。遺伝子発現ベクターは、被験者の体内で複製かつ発現が可能な様々な発現ベクターを利用することができる。例えば、ウイルスベクターが挙げられる。ウイルスベクターには、レトロウイルス（レンチウイルスを含む）、アデノウイルス、アデノ随伴ウイルス等に由来する種々のベクターが含まれる。 “Gene expression vector” refers to an expression unit that contains a gene or gene fragment in an expressible state and can control the expression of the gene or the like. In the present specification, the “expressible state” means that a ribosomal protein gene or the like is arranged in the downstream region under the control of a promoter. Therefore, expression of a ribosomal protein gene or the like is induced by the activation of the promoter. As the gene expression vector, various expression vectors that can be replicated and expressed in the body of a subject can be used. An example is a viral vector. Viral vectors include various vectors derived from retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, and the like.
本発明の組成物において有効成分は、複数種又は複数形態で含まれていてもよい。例えば、表１に記載のリボソームタンパク質において、大サブユニット構成タンパク質であるRpl26と小サブユニット構成タンパク質であるRps7を組み合わせた場合や、リボソームタンパク質であるRpl26とRpl26遺伝子を含む遺伝子発現ベクターを組み合わせた場合等が挙げられる。 In the composition of the present invention, the active ingredient may be contained in plural kinds or plural forms. For example, in the ribosomal proteins listed in Table 1, when Rpl26, which is a large subunit component protein, and Rps7, which is a small subunit component protein, are combined, or a gene expression vector containing Rpl26 and Rpl26 genes, which are ribosomal proteins, is combined. Cases.
組成物に含まれる有効成分の含有量は、有効成分の種類や形態（ペプチド、DNA又はmRNA）、組成物の剤形、並びに後述する溶媒や担体の種類によって異なる。したがって、それぞれの条件を勘案して適宜定めればよい。通常は、単回適用量の組成物に有効量の有効成分が含有されるように調整されている。しかし、有効成分の薬理効果を得る上で、被験者に組成物を大量に投与する必要がある場合、被験者の負担軽減のために数回に分割して投与することもできる。この場合、有効成分の量は、総合量で有効量を含んでいればよい。 The content of the active ingredient contained in the composition varies depending on the type and form of the active ingredient (peptide, DNA or mRNA), the dosage form of the composition, and the type of solvent or carrier described below. Therefore, it may be determined appropriately in consideration of each condition. Usually, it is adjusted so that an effective amount of an active ingredient is contained in a single application amount of the composition. However, when it is necessary to administer a large amount of the composition to the subject in order to obtain the pharmacological effect of the active ingredient, the composition can be divided into several times to reduce the burden on the subject. In this case, the amount of the active ingredient only needs to include the effective amount as a total amount.
本明細書において「有効量」とは、有効成分としての機能を発揮する上で必要な量であって、かつそれを適用する被験者に対して有害な副作用をほとんど又は全く付与しない量をいう。この有効量は、被験者の情報、適用経路、及び適用回数等の様々な条件によって変わり得る。本発明の組成物を医薬組成物として使用する場合、有効成分の含有量は、最終的には、医師又は薬剤師等の判断によって決定される。 In the present specification, the “effective amount” refers to an amount necessary for exerting a function as an active ingredient and giving little or no harmful side effects to a subject to which the function is applied. This effective amount may vary depending on various conditions such as subject information, application route, and number of applications. When using the composition of this invention as a pharmaceutical composition, content of an active ingredient is finally determined by judgment of a doctor or a pharmacist.
本明細書において「被験者」とは、ヒト個体であって、本発明の組成物の適用対象者をいう。好ましくはTDP-43プロテイノパチー罹患患者、例えば、ALS及び／又はFTLD罹患患者である。 As used herein, “subject” refers to a human individual who is an application target of the composition of the present invention. Preferred are patients with TDP-43 proteinopathy, such as patients with ALS and / or FTLD.
本明細書において、「被験者の情報」とは、被験者の特徴や状態に関する様々な情報である。例えば、年齢、体重、性別、全身の健康状態、疾患の有無、疾患の進行度や重症度、薬剤感受性、併用薬物の有無及び治療に対する耐性等が挙げられる。 In this specification, “subject information” refers to various information relating to the characteristics and state of the subject. Examples include age, weight, sex, general health, presence or absence of disease, progression or severity of disease, drug sensitivity, presence or absence of concomitant drugs, and resistance to treatment.
（２）溶媒
本発明の組成物は、必要に応じて薬学的に許容可能な溶媒中に溶解することができる。「薬学的に許容可能な溶媒」とは、製剤技術分野において通常使用する溶媒をいう。例えば、水若しくは水溶液、又は有機溶剤が挙げられる。水溶液には、例えば、生理食塩水、ブドウ糖又はその他の補助剤を含む等張液、リン酸塩緩衝液、酢酸ナトリウム緩衝液が挙げられる。補助剤には、例えば、D-ソルビトール、D-マンノース、D-マンニトール、塩化ナトリウム、その他にも低濃度の非イオン性界面活性剤、ポリオキシエチレンソルビタン脂肪酸エステル類等が挙げられる。有機溶剤には、エタノールが挙げられる。
(2) Solvent The composition of the present invention can be dissolved in a pharmaceutically acceptable solvent as necessary. “Pharmaceutically acceptable solvent” refers to a solvent usually used in the field of pharmaceutical technology. For example, water or an aqueous solution, or an organic solvent can be used. Examples of the aqueous solution include isotonic solutions containing physiological saline, glucose or other adjuvants, phosphate buffers, and sodium acetate buffers. Examples of the auxiliary agent include D-sorbitol, D-mannose, D-mannitol, sodium chloride, low concentration nonionic surfactants, polyoxyethylene sorbitan fatty acid esters, and the like. The organic solvent includes ethanol.
（３）担体
本発明の組成物は、必要に応じて薬学的に許容可能な担体を含むことができる。「薬学的に許容可能な担体」とは、製剤技術分野において通常使用する添加剤をいう。例えば、賦形剤、結合剤、崩壊剤、充填剤、乳化剤、流動添加調節剤、滑沢剤、ヒト血清アルブミン等が挙げられる。
(3) Carrier The composition of the present invention can contain a pharmaceutically acceptable carrier as required. “Pharmaceutically acceptable carrier” refers to an additive usually used in the field of pharmaceutical technology. Examples thereof include excipients, binders, disintegrants, fillers, emulsifiers, fluid addition regulators, lubricants, human serum albumin and the like.
賦形剤には、例えば、単糖、二糖類、シクロデキストリン及び多糖類のような糖、金属塩、クエン酸、酒石酸、グリシン、ポリエチレングリコール、プルロニック、カオリン、ケイ酸、又はそれらの組み合わせが挙げられる。 Excipients include, for example, sugars such as monosaccharides, disaccharides, cyclodextrins and polysaccharides, metal salts, citric acid, tartaric acid, glycine, polyethylene glycol, pluronic, kaolin, silicic acid, or combinations thereof. It is done.
結合剤には、例えば、植物デンプンを用いたデンプン糊、ペクチン、キサンタンガム、単シロップ、グルコース液、ゼラチン、トラガカント、メチルセルロース、ヒドロキシプロピルメチルセルロース、カルボキシメチルセルロースナトリウム、セラック、パラフィン、ポリビニルピロリドン又はそれらの組み合わせが挙げられる。 Examples of the binder include starch paste using plant starch, pectin, xanthan gum, simple syrup, glucose solution, gelatin, tragacanth, methylcellulose, hydroxypropylmethylcellulose, sodium carboxymethylcellulose, shellac, paraffin, polyvinylpyrrolidone, or combinations thereof. Can be mentioned.
崩壊剤としては、例えば、前記デンプンや、乳糖、カルボキシメチルデンプン、架橋ポリビニルピロリドン、アガー、ラミナラン末、炭酸水素ナトリウム、炭酸カルシウム、アルギン酸若しくはアルギン酸ナトリウム、ポリオキシエチレンソルビタン脂肪酸エステル、ラウリル硫酸ナトリウム、ステアリン酸モノグリセリド又はそれらの塩が挙げられる。 Examples of the disintegrant include the starch, lactose, carboxymethyl starch, crosslinked polyvinylpyrrolidone, agar, laminaran powder, sodium bicarbonate, calcium carbonate, alginic acid or sodium alginate, polyoxyethylene sorbitan fatty acid ester, sodium lauryl sulfate, stearin. Examples include acid monoglycerides or salts thereof.
充填剤としては、ワセリン、前記糖及び／又はリン酸カルシウムが例として挙げられる。 Examples of the filler include petrolatum, the sugar and / or calcium phosphate.
乳化剤としては、ソルビタン脂肪酸エステル、グリセリン脂肪酸エステル、ショ糖脂肪酸エステル、プロピレングリコール脂肪酸エステルが例として挙げられる。 Examples of the emulsifier include sorbitan fatty acid ester, glycerin fatty acid ester, sucrose fatty acid ester, and propylene glycol fatty acid ester.
流動添加調節剤及び滑沢剤としては、ケイ酸塩、タルク、ステアリン酸塩又はポリエチレングリコールが例として挙げられる。 Examples of the flow control agent and lubricant include silicate, talc, stearate or polyethylene glycol.
上記の他にも、必要であれば医薬組成物等において通常用いられる可溶化剤、懸濁剤、希釈剤、分散剤、界面活性剤、無痛化剤、安定剤、吸収促進剤、増量剤、付湿剤、保湿剤、湿潤剤、吸着剤、矯味矯臭剤、崩壊抑制剤、コーティング剤、着色剤、保存剤、防腐剤、抗酸化剤、香料、風味剤、甘味剤、緩衝剤、等張化剤等を適宜含むこともできる。 In addition to the above, solubilizers, suspending agents, diluents, dispersants, surfactants, soothing agents, stabilizers, absorption enhancers, extenders, which are usually used in pharmaceutical compositions, etc., if necessary Moisturizer, moisturizer, wetting agent, adsorbent, flavoring agent, disintegration inhibitor, coating agent, colorant, preservative, preservative, antioxidant, fragrance, flavoring agent, sweetener, buffering agent, isotonic An agent and the like can also be included as appropriate.
担体は、被験者体内で酵素等による前記有効成分の分解を回避又は抑制する他、製剤化や投与方法を容易にし、剤形及び薬効を維持するために用いられるものであり、必要に応じて適宜使用すればよい。 The carrier is used for avoiding or suppressing degradation of the active ingredient by an enzyme or the like in the body of the subject, facilitating formulation and administration methods, and maintaining the dosage form and drug efficacy. Use it.
３−２．剤形
本発明の組成物の剤形は、特に限定しない。被験者の体内で有効成分を失活させることなく目的の部位にまで送達できる形態であればよい。
3-2. Dosage Form The dosage form of the composition of the present invention is not particularly limited. Any form that can be delivered to the target site without inactivating the active ingredient in the body of the subject may be used.
具体的な剤形は、後述する適用方法によって異なる。適用方法は、非経口投与と経口投与に大別することができるので、それぞれの投与法に適した剤形にすればよい。 The specific dosage form varies depending on the application method described later. Since application methods can be broadly classified into parenteral administration and oral administration, dosage forms suitable for each administration method may be used.
例えば、投与方法が非経口投与であれば、好ましい剤形は、対象部位への直接投与又は循環系を介した全身投与が可能な液剤である。液剤の例としては、注射剤が挙げられる。注射剤は、前記賦形剤、乳化剤、懸濁剤、界面活性剤、安定剤、pH調節剤等と適宜組み合わせて、一般に認められた製薬実施に要求される単位用量形態で混和することによって製剤化することができる。 For example, if the administration method is parenteral administration, a preferred dosage form is a liquid that can be administered directly to the target site or systemically administered via the circulatory system. An example of the liquid agent is an injection. Injections are formulated by mixing in the unit dosage form generally required for pharmaceutical practice, in combination with the excipients, emulsifiers, suspensions, surfactants, stabilizers, pH regulators, etc. as appropriate. Can be
投与方法が経口投与であれば、好ましい剤形は、固形剤（錠剤、カプセル剤、ドロップ剤、トローチ剤を含む）、顆粒剤、粉剤、散剤、液剤（内用水剤、乳剤、シロップ剤を含む）が挙げられる。固形剤であれば、必要に応じて、当該技術分野で公知の剤皮を施した剤形、例えば、糖衣錠、ゼラチン被包錠、腸溶錠、フィルムコーティング錠、二重錠、多層錠にすることができる。 If the administration method is oral administration, preferred dosage forms include solid preparations (including tablets, capsules, drops, lozenges), granules, powders, powders, and liquids (internal solutions, emulsions, syrups). ). If it is a solid preparation, a dosage form with a coating known in the art, for example, a sugar-coated tablet, a gelatin-encapsulated tablet, an enteric tablet, a film-coated tablet, a double tablet, or a multilayer tablet, if necessary be able to.
なお、上記各剤形の具体的な形状、大きさについては、いずれもそれぞれの剤形において当該分野で公知の剤形の範囲内にあればよく、特に限定はしない。本発明の組成物の製造方法については、当該技術分野の常法に従って製剤化すればよい。 The specific shape and size of each dosage form are not particularly limited as long as each dosage form is within the range of dosage forms known in the art. What is necessary is just to formulate according to the conventional method of the said technical field about the manufacturing method of the composition of this invention.
３−３．適用方法
本発明の組成物の適用経路は、経口投与でも、非経口投与でもよい。経口投与法は、一般に全身投与であるが、非経口投与法は、さらに局所投与と全身投与に細分できる。局所投与には、例えば、筋肉内投与、皮下投与、組織投与、及び器官投与が該当し、全身投与には、例えば、静脈内投与（静注）及び動脈内投与が該当する。本発明の組成物を局所投与する場合には、注射等で中枢神経系、例えば、大脳（例えば、患部である前頭葉や側頭葉）、脳幹、脊髄に直接投与すればよい。また、全身投与する場合には、静注すればよい。投与量は、有効成分が奏効する上で有効な量であればよい。有効量は、前述のように被験者情報に応じて適宜選択されるが、適用対象が標準的な大人のヒト個体であれば、通常１回投与量当りの有効量は、50〜500 mg/m2となる。
3-3. Application Method The application route of the composition of the present invention may be oral administration or parenteral administration. Oral administration is generally systemic, but parenteral administration can be further subdivided into local and systemic administration. Local administration includes, for example, intramuscular administration, subcutaneous administration, tissue administration, and organ administration, and systemic administration includes, for example, intravenous administration (intravenous injection) and intraarterial administration. When the composition of the present invention is locally administered, it may be administered directly to the central nervous system, for example, the cerebrum (for example, the frontal lobe or temporal lobe, which is an affected area), the brain stem, or the spinal cord by injection or the like. In addition, intravenous administration may be used for systemic administration. The dose may be an amount that is effective for the effective ingredient to be effective. The effective dose is appropriately selected according to the subject information as described above. However, if the subject of application is a standard adult human individual, the effective dose per dose is usually 50 to 500 mg / m. 2
＜実施例１：TDP-43による神経突起内輸送標的mRNAの同定＞
（目的）
TDP-43によって神経突起内に輸送される標的mRNAを単離し、同定した。
（方法）
（１）TDP-43 shRNA発現レンチウイルスベクターの構築
TDP-43 shRNA1をコードする配列番号９８で示す塩基配列からなるDNA、TDP-43 shRNA2をコードする配列番号９９で示す塩基配列からなるDNA、及び対照用shRNAをコードする配列番号１００で示す塩基配列からなるDNAを、U6 snRNA遺伝子のプロモーター下に連結し、それぞれをレンチウイルス用発現プラスミドpLKO.1-puro（シグマアルドリッチ社）内に挿入して、2種類のTDP-43 shRNA発現レンチウイルスベクター（pTDP-43 shRNA1及びpTDP-43 shRNA2）、及び対照用shRNA発現レンチウイルスベクター（Cont shRNA）を構築した。
<Example 1: Identification of neurite transport target mRNA by TDP-43>
(the purpose)
The target mRNA transported into neurites by TDP-43 was isolated and identified.
(Method)
(1) Construction of TDP-43 shRNA expression lentiviral vector
DNA consisting of the base sequence shown in SEQ ID NO: 98 encoding TDP-43 shRNA1, DNA consisting of the base sequence shown in SEQ ID NO: 99 encoding TDP-43 shRNA2, and base sequence shown in SEQ ID NO: 100 encoding shRNA for control Are ligated under the promoter of U6 snRNA gene, each of which is inserted into lentiviral expression plasmid pLKO.1-puro (Sigma Aldrich), and two types of TDP-43 shRNA expression lentiviral vectors ( pTDP-43 shRNA1 and pTDP-43 shRNA2) and a control shRNA expression lentiviral vector (Cont shRNA) were constructed.
（２)神経細胞の培養
神経突起単離用の神経細胞には、マウスの大脳皮質神経を用いた。胎生15日目のマウス胎児より大脳皮質神経細胞を採取し、DMEM+10% FBS+Penicillin/Streptomycin培地（「培地A」とする）（和光純薬工業）中に浸漬した後述のインサートチャンバー培養装置における多孔性メンブレンの上部に播種した後、5％ CO2存在下において37℃にて一晩培養した（1日目）。
(2) Nerve cell culture Mouse cerebral cortical nerves were used as neurite isolation neurons. Cerebral cortical neurons collected from embryonic day 15 mouse embryos and immersed in DMEM + 10% FBS + Penicillin / Streptomycin medium (referred to as “medium A”) (Wako Pure Chemical Industries) After seeding on the upper part of the porous membrane, the cells were cultured overnight at 37 ° C. in the presence of 5% CO 2 (1st day).
培養2日目以降はNeuro Medium+2% NeuroBrew21+Penicillin/Streptomycin+1mM sodium pyruvate+27.5μM 2-mercaptoethanol（「培地B」とする）（ミルテニーバイオテク社）で、5％ CO2存在下において37℃にて1週間培養した。 From the 2nd day onward, Neuro Medium + 2% NeuroBrew21 + Penicillin / Streptomycin + 1 mM sodium pyruvate + 27.5 μM 2-mercaptoethanol (referred to as “medium B”) (Miltenyi Biotech), 37% in the presence of 5% CO 2 Incubated for 1 week at ℃.
（３）TDP-43 shRNA発現レンチウイルスベクターの神経細胞内導入
TDP-43の発現が抑制された神経細胞は、構築したTDP-43 shRNA発現レンチウイルスベクターを神経細胞内に導入して作製した。
(3) Introduction of TDP-43 shRNA expressing lentiviral vector into neurons
The nerve cell in which the expression of TDP-43 was suppressed was prepared by introducing the constructed TDP-43 shRNA expression lentiviral vector into the nerve cell.
まず、30μgのpTDP-43 shRNA1、pTDP-43 shRNA2又はCont shRNA、22.5μgのパッケージングプラスミドdelta8.9（Addgene社）、及び7.5μgのエンベローププラスミドVSV-G（Addgene社）を混合して、これを「発現ベクター混合液」とした。293FT細胞を播種した15cmディッシュの培地Aに前述の発現ベクター混合液を加えて、リン酸カルシウム法を用いて細胞内に導入した。5％ CO2存在下において37℃にて一晩培養した後、培地Aを交換し、その24時間後と48時間後の両時間において培地を回収した。両培地を混合したものを「ウイルス液」として調製した。 First, mix 30 μg of pTDP-43 shRNA1, pTDP-43 shRNA2 or Cont shRNA, 22.5 μg of packaging plasmid delta8.9 (Addgene), and 7.5 μg of envelope plasmid VSV-G (Addgene). Was used as an “expression vector mixture”. The above-mentioned expression vector mixture was added to 15 cm dish of medium A seeded with 293FT cells, and introduced into the cells using the calcium phosphate method. After overnight culture at 37 ° C. in the presence of 5% CO 2 , the medium A was exchanged, and the medium was collected at both 24 and 48 hours. A mixture of both media was prepared as a “virus solution”.
続いて、上記「（２）神経細胞の培養」における培養開始翌日（上記2日目）の培地交換前に、上記ウイルス液を培地Aに加えて、5％ CO2存在下において37℃で6時間培養してレンチウイルスを感染させた。ここで、pTDP-43 shRNA1とpTDP-43 shRNA2を感染させた細胞をそれぞれ「TDP-sh1」及び「TDP-sh2」とした。またCont shRNAを感染させた細胞は「Cont-sh」とした。その後、培地を培地Bに交換して、5％ CO2存在下で37℃にて、さらに1週間培養を続けた後、神経突起分画を採取した。 Subsequently, the virus solution is added to the medium A before the medium exchange on the next day (the second day) in the above “(2) Nerve cell culture”, and is performed at 37 ° C. in the presence of 5% CO 2. Incubate for hours to infect lentivirus. Here, cells infected with pTDP-43 shRNA1 and pTDP-43 shRNA2 were designated as “TDP-sh1” and “TDP-sh2”, respectively. The cells infected with Cont shRNA were designated as “Cont-sh”. Thereafter, the medium was changed to medium B, and the culture was continued at 37 ° C. in the presence of 5% CO 2 for another week, and then the neurite fraction was collected.
（４）神経突起の採取
神経細胞から神経突起と単離するために、図１で示すインサートチャンバー培養装置を用いた。この培養装置は、底面に1μm径の多孔性メンブレン（図１−003）を張ったインサートチャンバー（図１−001）を3mLの培地A（図１−004）を入れた6ウェルプレート（図１−002）内に挿入した構成を有する。
(4) Collection of neurites In order to isolate neurites from nerve cells, the insert chamber culture apparatus shown in FIG. 1 was used. This culture apparatus has an insert chamber (FIG. 1-001) with a 1 μm-diameter porous membrane (FIG. 1-003) on the bottom and a 6-well plate containing 3 mL of medium A (FIG. 1-004) (FIG. 1). -002).
上記「（２）神経細胞の培養」及び「（３）TDP-43 shRNA発現レンチウイルスベクターの神経細胞内導入」に記載のようにTDP-sh1、TDP-sh2、及びCont-shを1週間培養後、それぞれのインサートチャンバーの多孔性メンブレン下面より突出した神経突起（図１−006）を各神経突起分画として回収した。 TDP-sh1, TDP-sh2, and Cont-sh are cultured for 1 week as described in “(2) Nerve cell culture” and “(3) Introduction of TDP-43 shRNA-expressing lentiviral vector into neurons”. Thereafter, neurites (FIG. 1-006) protruding from the lower surface of the porous membrane of each insert chamber were collected as each neurite fraction.
（mRNAの抽出及びマイクロアレイ解析）
回収したTDP-sh1及びTDP-sh2、並びにCont-shの各神経突起分画よりTRIZOL（Thermo Fisher Scientific社）を用いてtotal RNAを抽出した。3D-Gene（登録商標）（東レ社）によるマイクロアレイ解析によりCont-shと比較したときにTDP-sh1及びTDP-sh2の両者において有意に減少しているmRNAを標的mRNA候補として同定した。
(MRNA extraction and microarray analysis)
Total RNA was extracted from the recovered TDP-sh1 and TDP-sh2 and Cont-sh neurite fractions using TRIZOL (Thermo Fisher Scientific). By the microarray analysis by 3D-Gene (registered trademark) (Toray Industries, Inc.), mRNA significantly decreased in both TDP-sh1 and TDP-sh2 when compared with Cont-sh was identified as a target mRNA candidate.
（結果）
TDP-sh1及びTDP-sh2の両者で減少するmRNAが、約1000遺伝子単離された。gene ontology解析によりCont-shに対して有意に減少した遺伝子として、表１に挙げる細胞質型リボソームタンパク質が単離された。なお、表１に示すリボソームタンパク質のアミノ酸配列及びその遺伝子の塩基配列は、ヒトオルソログの配列である。
(result)
About 1000 genes were isolated from mRNA that decreased in both TDP-sh1 and TDP-sh2. The cytoplasmic ribosomal proteins listed in Table 1 were isolated as genes that were significantly reduced with respect to Cont-sh by gene ontology analysis. The amino acid sequence of the ribosomal protein and the base sequence of the gene shown in Table 1 are human orthologue sequences.
＜実施例２：輸送標的遺伝子候補の過剰発現による神経突起伸長障害の救済効果＞
（目的）
TDP-43 shRNAによりTDP-43の発現が抑制された大脳皮質神経細胞（TDP-sh細胞）では、神経突起の伸長障害がみられる（図２−１A）。そこで、TDP-sh細胞において、リボソームタンパク質遺伝子を過剰発現させたときの救済効果について検証した。
（方法）
0.5mLの培地Aを入れた24ウェルプレートにマウス胎児大脳皮質神経細胞を播種し、5％ CO2存在下で37℃にて一晩培養した。培養後、実施例１で構築したpTDP-43 shRNA1、実施例１で単離された各標的mRNA候補のマウスリボソームタンパク質遺伝子発現するレンチウイルスベクター（CSII-CMV-Rp-IRES2-Venus）を2種同時に6時間感染させた後、培地を培地Bに交換し、5％ CO2存在下で37℃にて6日間培養した。なお、レンチウイルスベクターCSII-CMV-Rp-IRES2-Venusは、CMVプロモーター下に連結した標的mRNA候補のマウスリボソームタンパク質遺伝子とIRES配列下に蛍光タンパク質Venusの遺伝子を連結した構成を有し、標的マウスリボソームタンパク質とVenusタンパク質を同時に発現することができる。標的mRNA候補のマウスリボソームタンパク質遺伝子は、大サブユニットから任意に選択したmRpl26遺伝子（配列番号９５）、及び小サブユニットから任意に選択したmRps7遺伝子（配列番号９６）を用いた。培養後に蛍光顕微鏡で神経突起を観察した。
<Example 2: Relief effect of neurite outgrowth disorder by overexpression of transport target gene candidate>
(the purpose)
In cerebral cortical neurons (TDP-sh cells) in which TDP-43 expression is suppressed by TDP-43 shRNA, neurite outgrowth is impaired (FIG. 2-1A). Therefore, the rescue effect when the ribosomal protein gene was overexpressed in TDP-sh cells was examined.
(Method)
Mouse embryonic cerebral cortical neurons were seeded in a 24-well plate containing 0.5 mL of medium A and cultured overnight at 37 ° C. in the presence of 5% CO 2 . After culturing, two types of pTDP-43 shRNA1 constructed in Example 1 and lentiviral vectors (CSII-CMV-Rp-IRES2-Venus) expressing mouse ribosomal protein genes of each target mRNA candidate isolated in Example 1 were used. After simultaneously infecting for 6 hours, the medium was changed to medium B and cultured at 37 ° C. for 6 days in the presence of 5% CO 2 . The lentiviral vector CSII-CMV-Rp-IRES2-Venus has a structure in which the target mRNA candidate mouse ribosomal protein gene linked under the CMV promoter and the fluorescent protein Venus gene linked under the IRES sequence are linked to the target mouse. Ribosomal and Venus proteins can be expressed simultaneously. As the mouse ribosomal protein gene of the target mRNA candidate, mRpl26 gene (SEQ ID NO: 95) arbitrarily selected from the large subunit and mRps7 gene (SEQ ID NO: 96) arbitrarily selected from the small subunit were used. The neurites were observed with a fluorescence microscope after incubation.
また、上記マウス胎児大脳皮質神経細胞の培養開始から3日後にpTDP-43 shRNA1、及びCMVプロモーター下にmRpl26遺伝子、mRpl41遺伝子（配列番号９７）、mRps7遺伝子、又はヒトTDP-43遺伝子（配列番号２）をそれぞれ発現するプラスミド（pcDNA3.1-CMV-mRpl26、pcDNA3.1-CMV-mRpl41、pcDNA3.1-CMV-mRps7、及びpcDNA3.1-CMV-hTDP-43）、及びCAGプロモーター下にEGFPを発現するプラスミド（pCAG2-EGFP）の3種発現ベクターをリポフェクタミン（登録商標）2000（Thermo Fisher Scientific社）により同時に導入した後、5％ CO2存在下で37℃にて2日間培養した。対照用として、Cont shRNA、挿入配列を含まないpcDNA3.1及びpCAG2-EGFP、及びpTDP-43 shRNA1、pcDNA3.1及びpCAG2-EGFPの3種発現ベクターを上記と同様に同時導入して、2日間培養した。
培養後に各神経細胞において最も長い神経突起の長さを定量した。
In addition, 3 days after the start of the culture of the mouse embryonic cerebral cortex neurons, pTDP-43 shRNA1, and mRpl26 gene, mRpl41 gene (SEQ ID NO: 97), mRps7 gene, or human TDP-43 gene (SEQ ID NO: 2) under the CMV promoter. ) Plasmids (pcDNA3.1-CMV-mRpl26, pcDNA3.1-CMV-mRpl41, pcDNA3.1-CMV-mRps7, and pcDNA3.1-CMV-hTDP-43), and EGFP under the CAG promoter. Three expression vectors for the expression plasmid (pCAG2-EGFP) were simultaneously introduced by Lipofectamine (registered trademark) 2000 (Thermo Fisher Scientific), and then cultured at 37 ° C. in the presence of 5
The longest neurite length was quantified in each neuron after culture.
（結果）
神経突起の伸長を示す蛍光図を図２−１に、また神経突起の長さの定量図を図２−２に示す。
(result)
A fluorescence diagram showing neurite outgrowth is shown in FIG. 2-1, and a quantitative diagram of neurite length is shown in FIG. 2-2.
図２−１Aで示すようにTDP-sh細胞では大脳皮質神経細胞内のTDP-43の発現をTDP-43 shRNAにより抑制した結果、神経突起の伸長が著しく阻害された。しかし、B及びCで示すように、TDP-43 shRNAの存在下であっても実施例１で単離されたTDP-43の神経突起輸送標的mRNA候補であるRpl26遺伝子、又はRps7遺伝子を過剰発現させた場合には、神経突起の伸長が有意に改善することが示された。 As shown in FIG. 2-1A, in TDP-sh cells, TDP-43 expression in cerebral cortical neurons was suppressed by TDP-43 shRNA. As a result, neurite outgrowth was significantly inhibited. However, as shown in B and C, even in the presence of TDP-43 shRNA, overexpression of Rpl26 gene or Rps7 gene, which is a candidate neurite transport target mRNA of TDP-43 isolated in Example 1 When done, it was shown that neurite outgrowth improved significantly.
また、図２−２で示すように、リポフェクタミン（登録商標）2000（Thermo Fisher Scientific社）を用いた場合もRpl26遺伝子、Rpl41遺伝子及びRps7遺伝子の過剰発現により、神経突起の伸長がいずれも対照用shRNA処理の神経細胞（Cont-sh）と同程度まで改善した。またTDP-43 shRNAによる神経突起の伸長障害も、TDP-43遺伝子やLa遺伝子の過剰発現によって有意に救済されることが明らかとなった。 In addition, as shown in Fig. 2-2, when Lipofectamine (registered trademark) 2000 (Thermo Fisher Scientific) was used, neurite outgrowth was all for control due to overexpression of Rpl26 gene, Rpl41 gene and Rps7 gene. It was improved to the same level as shRNA-treated neurons (Cont-sh). In addition, it was revealed that neurite outgrowth damage caused by TDP-43 shRNA is significantly rescued by overexpression of TDP-43 gene and La gene.
以上の結果は、TDP-43の輸送標的遺伝子がリボソームタンパク質遺伝子であること、そして大サブユニット又は小サブユニットにかかわらず、リボソームタンパク質であれば1遺伝子であっても、その過剰発現によりTDP-43の発現低下によって生じる神経突起の伸長障害を救済できることが明らかとなった。 The above results indicate that the TDP-43 transport target gene is a ribosomal protein gene, and even if it is a single ribosomal protein regardless of the large subunit or small subunit, overexpression of TDP-43 It became clear that the neurite outgrowth disorder caused by the reduced expression of 43 can be rescued.
＜実施例３：リボソームタンパク質遺伝子の過剰発現によるTDP-43変異型ショウジョウバエの複眼変性の救済効果＞
（目的）
リボソームタンパク質遺伝子の過剰発現が、TDP-43の機能障害による神経突起伸長障害以外の表現型異常を改善できるか否かを検証した。
（方法）
TDP-43の機能障害モデル生物として、キイロショウジョウバエ（Drosophila melanogaster）のGLA4-UAS系（Brand A. H. ＆ Perrimon N., 1993, Development, 118: 401）によるTDP-43過剰発現株を用いた。UAS-TDP-43株は、GatewayデスティネーションベクターであるpUAST-DESTに挿入された全長ヒトTDP-43 cDNAをハエembryoにインジェクションすることにより得た（BestGene Inc.により作成した）。
<Example 3: Relief effect of compound eye degeneration of TDP-43 mutant Drosophila by overexpression of ribosomal protein gene>
(the purpose)
We examined whether overexpression of the ribosomal protein gene can improve phenotypic abnormalities other than neurite outgrowth disorder caused by TDP-43 dysfunction.
(Method)
As a TDP-43 dysfunction model organism, a strain overexpressing TDP-43 by Drosophila melanogaster GLA4-UAS system (Brand AH & Perrimon N., 1993, Development, 118: 401) was used. The UAS-TDP-43 strain was obtained by injecting a full-length human TDP-43 cDNA inserted into the gateway destination vector pUAST-DEST into a fly embryo (made by BestGene Inc.).
TDP-43は、細胞内の発現量、存在量、局在性が厳密に制御されている。それ故に発現抑制や過剰発現のように通常範囲を逸脱した場合には、個体に何らかの変化が現れ得る。キイロショウジョウバエにおいてもTDP-43遺伝子の過剰発現個体や発現抑制個体では、複眼を構成する一部の個眼が壊死して黒い斑点状となるnecrotic patch（壊死斑点）が現れる複眼変性が認められる。そこで、キイロショウジョウバエのTDP-43過剰発現個体でリボソームタンパク質遺伝子を過剰発現させたときのnecrotic patchの出現率を検証した。 In TDP-43, the expression level, abundance, and localization in cells are strictly controlled. Therefore, when the expression deviates from the normal range, such as expression suppression or overexpression, some change may appear in the individual. Even in Drosophila melanogaster, compound degeneration in which TDP-43 gene overexpression and suppression of expression occur necrotic patches (necrotic spots) in which part of the compound eye is necrotic and necrotic. Therefore, we examined the appearance rate of necrotic patch when overexpression of ribosomal protein gene in TDP-43 overexpressing individuals of Drosophila melanogaster.
まず、キイロショウジョウバエのリボソームタンパク質遺伝子であるdRpl26遺伝子とdRpl41遺伝子について、前述のUAS-hTDP-43株と同様にしてUAS系株（UAS-dRpl26株及びUAS-Rpl41株）を作製した。 First, UAS strains (UAS-dRpl26 strain and UAS-Rpl41 strain) were prepared for the dRpl26 gene and dRpl41 gene, which are ribosomal protein genes of Drosophila melanogaster, in the same manner as the UAS-hTDP-43 strain described above.
続いて、gmr-GAL4ドライバー株（gmr-GAL4/+）とUAS-hTDP-43株（UAS-hTDP-43/+）を交配し、TDP-43過剰発現株を作出した。さらに得られたTDP-43過剰発現株にUAS-dRpl26株（UAS-dRpl26/+）、UAS-dRpl41株（UAS-dRpl41/+）、又はUAS-EGFP株（UAS-EGFP/+）を交配させることにより、複眼でhTDP-43遺伝子とdRpl26遺伝子、hTDP-43遺伝子とdRpl41遺伝子、又は対照用のhTDP-43遺伝子とEGFP遺伝子を過剰発現するキイロショウジョウバエ（それぞれhTDP-43/dRpl26 o/e株、hTDP-43/dRpl41 o/e株、及びhTDP-43/EGFP o/e株とする）を作出した。飼育及び交配は25℃で行い、複眼変性の程度は羽化後1日の時点で光学実体顕微鏡を用いて観察した。 Subsequently, the gmr-GAL4 driver strain (gmr-GAL4 / +) and the UAS-hTDP-43 strain (UAS-hTDP-43 / +) were crossed to produce a TDP-43 overexpressing strain. Furthermore, UAS-dRpl26 strain (UAS-dRpl26 / +), UAS-dRpl41 strain (UAS-dRpl41 / +), or UAS-EGFP strain (UAS-EGFP / +) is crossed with the obtained TDP-43 overexpression strain Drosophila overexpressing hTDP-43 gene and dRpl26 gene, hTDP-43 gene and dRpl41 gene, or control hTDP-43 gene and EGFP gene in compound eyes (hTDP-43 / dRpl26 o / e strain, respectively) hTDP-43 / dRpl41 o / e strain and hTDP-43 / EGFP o / e strain). Breeding and mating were performed at 25 ° C., and the degree of compound eye degeneration was observed using an optical stereomicroscope one day after emergence.
（結果）
図３−１にキイロショウジョウバエ成虫のTDP-43過剰発現株において、さらにリボソームタンパク質を過剰発現させた時の各遺伝子型の代表的な2個体の複眼を示した図を示す。対照用のhTDP-43/EGFP o/e株（A及びB）で出現していたnecrotic patchは、hTDP-43/dRpl26 o/e株（C及びD）及びhTDP-43/dRpl41 o/e株（E及びF）では顕著に改善された。
(result)
FIG. 3-1 shows a compound eye of two representative individuals of each genotype when the ribosomal protein is further overexpressed in the TDP-43 overexpression strain of Drosophila melanogaster. The necrotic patches that appeared in the control hTDP-43 / EGFP o / e strains (A and B) were hTDP-43 / dRpl26 o / e strains (C and D) and hTDP-43 / dRpl41 o / e strains. (E and F) are markedly improved.
図３−２では、各遺伝子型10〜15個体の複眼におけるnecrotic patchを有する個体の比率を示している。necrotic patchの有無は、図３−１Bの矢印で示された4つのnecrotic patchのうち星印の付いた最も小さいものを基準として、これよりも大きいものが1つ以上認められた場合には「necrotic patchアリ」と判定した。対照用のhTDP-43/EGFP o/e株（A及びB）に対して、Rpl26遺伝子又はRpl41遺伝子を過剰発現させたhTDP-43/dRpl26 o/e株（C及びD）及びhTDP-43/dRpl41 o/e株（E及びF）では、necrotic patchの出現比率が低下し、複眼変性が軽減された。 FIG. 3-2 shows the ratio of individuals having necrotic patches in the compound eyes of 10 to 15 genotypes. The presence or absence of a necrotic patch is determined when one or more of the four necrotic patches indicated by the arrows in FIG. Necrotic patch ants ”. The hTDP-43 / dRpl26 o / e strain (C and D) overexpressing the Rpl26 gene or the Rpl41 gene (C and D) and the hTDP-43 / with respect to the control hTDP-43 / EGFP o / e strain (A and B) In the dRpl41 o / e strains (E and F), the appearance ratio of necrotic patch decreased and compound eye degeneration was reduced.
以上の結果は、TDP-43の機能障害によって現れる表現型の異常は、神経突起の伸長障害だけでなく、他の表現型であってもリボソームタンパク質遺伝子の過剰発現により救済できることを示している。これは、リボソームタンパク質やそれをコードする遺伝子がTDP-43プロテイノパチー治療用組成物の有効成分となり得ることを示唆している。 The above results indicate that phenotypic abnormalities caused by TDP-43 dysfunction can be rescued not only by neurite outgrowth but also by other phenotypes by overexpression of ribosomal protein genes. This suggests that the ribosomal protein and the gene encoding it can be an active ingredient in the composition for treating TDP-43 proteinopathy.
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