JP6516196B2 - マイクロrna‐328アンチセンス組成物及び治療用途 - Google Patents
マイクロrna‐328アンチセンス組成物及び治療用途 Download PDFInfo
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- JP6516196B2 JP6516196B2 JP2017567217A JP2017567217A JP6516196B2 JP 6516196 B2 JP6516196 B2 JP 6516196B2 JP 2017567217 A JP2017567217 A JP 2017567217A JP 2017567217 A JP2017567217 A JP 2017567217A JP 6516196 B2 JP6516196 B2 JP 6516196B2
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- A61K31/7088—Compounds having three or more nucleosides or nucleotides
- A61K31/711—Natural deoxyribonucleic acids, i.e. containing only 2'-deoxyriboses attached to adenine, guanine, cytosine or thymine and having 3'-5' phosphodiester links
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- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/7088—Compounds having three or more nucleosides or nucleotides
- A61K31/712—Nucleic acids or oligonucleotides having modified sugars, i.e. other than ribose or 2'-deoxyribose
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- C12N2310/10—Type of nucleic acid
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- C12N2310/141—MicroRNAs, miRNAs
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Description
接触できないRNAと呼ばれることもある「ロックド核酸」(LNA)は修飾RNAヌクレオチドである。LNAヌクレオチドのリボース部分は2’酸素と4’炭素とを結合する付加的な架橋で修飾される。架橋は、A形態の二本鎖において見られることも多い3’‐エンド(ノース)構造のリボースを「ロック」する。LNAヌクレオチドは、必要に応じてオリゴヌクレオチド中のDNA又はRNA残基と混合することが可能である。
*:ホスホジエステル結合をホスホロチオエート結合で置換したことを示す。
^は、配列番号18〜23が、表中の「+」や「*」で示しているそれぞれのLNA修飾及びPS修飾したそれらの対応物の天然(非修飾)オリゴヌクレオチドの配列であることを示す。
DNAヌクレオチドから成るアンチセンスmiR‐328オリゴヌクレオチドを成熟ヒトmiR‐328及びヒトpre‐328配列に従って設計した。メーカー説明書に従って、Dr.Oligo192と呼ばれるDNA/RNA合成機(Biolytic Lab Performance社)を用いてDNAアンチセンスオリゴヌクレオチドを合成した。全てのデオキシリボヌクレオチドを含む15〜30量体のアンチセンスオリゴヌクレオチドを作成した。生成物をHPLCで精製した。アンチセンスオリゴヌクレオチドの合成及び精製はGenomics BioSci&Tech社(台湾)により行った。DNA15量体〜30量体の配列を表1に示す。
17〜22量体の範囲のLNA修飾及びPS結合修飾アンチセンスオリゴヌクレオチドを成熟ヒトmiR‐328配列及びギャップマーの原理に従って設計した;修飾配列及び非修飾形態を表2に示す。この設計に従って、17〜22量体のLNA修飾及びPS結合修飾アンチセンス配列を合成し、Exiqon社(デンマーク)により精製した。
細胞培養、処理及びトランスフェクション
「ARPE‐19」と呼ばれるRPE細胞株をダルベッコ改変イーグル培地(DMEM)/F12培地で培養した。細胞系は、95%空気/5%二酸化炭素(CO2)の加湿雰囲気下、37℃で1%ペニシリン/ストレプトマイシン及び10%熱不活性化ウシ胎仔血清(FBS)と共に培養した。トランスフェクション実験を行うため、細胞を1×105個/ウェルの密度で12ウェルプレートに播種した。ウェル中で70%コンフルエントに達した後、メーカー説明書に従ってアンチセンスオリゴヌクレオチド(濃度30、50及び100nM)をLipofectamine2000(Invitrogen社)でトランスフェクトした。インキュベーションの24時間後、細胞を次の試験のために溶解した。
メーカー説明書に従って、Trizolを用いて培養細胞から全RNAを抽出した。A260/A280の読み取りを利用してRNA純度を調べた。初めに、以下の手順によりmiR‐328をmiR‐328cDNAに逆転写した:miR‐328特異的プライマー及びMultiScribe逆転写酵素キット(Applied Biosystems社)により5ngのRNAを逆転写した。miR‐328発現を測定するために、メーカー説明書(Applied Biosystems社)に従って、miR‐328特異的プローブを備えたABI7500リアルタイムPCR装置(Applied Biosystems社)において定量的リアルタイムPCRを実施した。定量的リアルタイムPCRによりmiR−328cDNAレベルを測定することで、アンチセンスオリゴヌクレオチドによる阻害効果を評価した。miR‐328の発現レベルを、内部標準として小さい非コード核RNA U6の発現レベルへと正規化した。
a.DNAアンチセンスオリゴヌクレオチド
RPE細胞におけるmiR‐328の発現に対する阻害効果について、実施例1に従って調製した16個の長さ15〜30量体のDNAアンチセンスオリゴヌクレオチドを調べた。相対的miR‐328発現レベルを図1に示す。DNA16量体及びDNA17量体のみがmiR‐328発現を阻害した。DNA16量体の阻害効果は、30、50及び100nMの濃度で39%、35%及び49%であった。DNA17量体では、その阻害効果は、30、50及び100nMの濃度で43%、41%及び48%であった。残りのアンチセンスオリゴヌクレオチド(15量体及び18〜30量体)はmiR‐328発現に対する阻害効果を示さなかった。
RPE細胞におけるmiR‐328発現に対する阻害効果について、名称がLNA401〜LNA406である6つのLNA修飾アンチセンス配列を試験した。相対的なmiR‐328発現レベルを図2に示す。LNA403、LNA404及びLNA405のみがmiR‐328発現を阻害した。LNA403の阻害効果は、30、50及び100nMの濃度でそれぞれ97%、97%及び90%であった。LNA404及びLNA405の阻害効果は、30、50及び100nMの濃度でそれぞれ98%、98%及び99%と、同じであった。残りのLNAオリゴヌクレオチドはmiR‐328発現に対して阻害効果を示さなかった。
動物モデル
21日齢のC57BL/6Jマウスを台湾のNational Laboratory Animal Centerから購入した。動物実験は全て、ARVO Statement for the Use of Animals in Ophthalmic and Vision Researchに準拠した。近視誘発の手順を簡単に以下のように説明する:23日齢のマウスの右眼を近視(すなわち近眼)を誘発するために覆い隠し、左眼は覆い隠すことはしなかった。その後、30日目、37日目及び44日目に、近視誘発マウスの右眼を30μLの生理食塩水(すなわち対照群)、1μMのDNA16量体、DNA17量体、LNA403、LNA404又はLNA405で処置した。51日目にマウスを屠殺し、眼を回収した。単離した眼を解剖顕微鏡下で撮影し、軸長を画像Jにより測定した。近視(近眼)は、近視の主要な病理学的変化である軸長の伸長を引き起こす。同じマウスの覆い隠した右眼と覆い隠していない左眼との軸長差(すなわちΔ軸長)は近視の重症度を示す。結果を表3及び図3に要約する。統計的評価はマンホイットニーU検定により行った。p値<0.05である分析は有意であると考える。
DNA16量体及びDNA17量体で処置したマウスの平均Δ軸長は、対照動物より統計的に有意に小さかった。DNA16量体及びDNA17量体では、Δ軸長は対照群と比較して0.077mm及び0.065mm有意に減少した。結果から、DNA16量体及びDNA17量体はマウス近視の治療に有効であることが分かった。
LNA403〜405で処置したマウスの平均Δ軸長は、対照動物より小さかった。LNA403ではΔ軸長は対照群と比較して0.054mm減少し、LNA404でははΔ軸長は0.025mm減少し、LNA405ではΔ軸長は0.035mm減少した。しかし、統計的有意性を示している(p値<0.05)のはLNA403のみである。結果から、LNA403はマウス近視の治療に有効であることが分かった。
動物モデル
21日齢のC57BL/6Jマウスを台湾のNational Laboratory Animal Centerから購入した。動物実験は全て、ARVO Statement for the Use of Animals in Ophthalmic and Vision Researchに準拠した。近視誘発の手順を簡単に以下のように説明する:23日齢のマウスの右眼を近視(すなわち近眼)を誘発するために4週間覆い隠し、左眼は覆い隠すことはしなかった。その後、30日目、37日目及び44日目に、近視誘発マウスの右眼を30μLの生理食塩水(すなわち陰性対照群)、1%のアトロピン(すなわち陽性対照群)、1μMのDNA16量体又はLNA403で処置した。51日目にマウスを屠殺し、眼を回収した。単離した眼を解剖顕微鏡下で撮影し、軸長を画像J及び自動測定のための専用ソフトウェアにより測定した。近視(近眼)は、近視の主要な病理学的変化である軸長の伸長を引き起こす。同じマウスの覆い隠した右眼と覆い隠していない左眼との軸長差(すなわちΔ軸長)は近視の重症度を示す。結果を表4及び図4に要約する。統計的評価は、マンホイットニーU検定により行った。p値<0.05である分析は有意であると考える。
本発明者らのDNA16量体の結果を確認するためにCROを利用した。
近視誘発の手順は以下のとおりである:21日齢のC57BL/6Jマウスを台湾のNational Laboratory Animal Centerから購入した。動物実験は全て、ARVO Statement for the Use of Animals in Ophthalmic and Vision Researchに準拠した。近視誘発の手順を簡単に以下のように説明する:23日齢のマウスの右眼を近視を誘発するために4週間覆い隠し、左眼は覆い隠すことはしなかった。その後、30日目、37日目及び44日目に、近視誘発マウスの右眼を30μLの生理食塩水(すなわち陰性対照群)、1%のアトロピン(すなわち陽性対照群)、10nM、100nM及び1μMのDNA16量体で処置した。51日目にマウスを屠殺し、眼を回収した。単離した眼を解剖顕微鏡下で撮影し、軸長を画像J及び自動測定のための専用ソフトウェアにより測定した。近視(近眼)は、近視の主要な病理学的変化である軸長の伸長を引き起こす。同じマウスの覆い隠した右眼と覆い隠していない左眼との軸長差(すなわちΔ軸長)は近視の重症度を示す。
動物モデル
近視誘発の手順:3日齢の着色ウサギを台湾のDA‐ZONG畜産場から購入した。動物実験は全て、ARVO Statement for the Use of Animals in Ophthalmic and Vision Researchに準拠した。7日齢で、ウサギの右眼を8週間覆い隠して近視を誘発し、左眼は覆い隠さないままにした。21日目から両眼の軸長(AXL)を週に1回、A‐scan(Sonomed(登録商標)、PACSCAN 300A、米国)により測定した。近視は、35日目に右眼AXLが左眼AXLより0.2mm以上長い状態であると定義した。ウサギがこの定義した基準に達すると、このような近視のウサギの右眼を、20μLの生理食塩水(すなわち対照群)又は10μMもしくは50μMのDNA16量体(20μL)で35日目から63日目まで1日おきに処置した。61日目に測定した最後のAXLを用いて右AXLと左AXLとの差(すなわちΔAXL)を求め、計算した。治療効果がない場合、ΔAXLは正の値となる。
Claims (8)
- 配列番号3又は配列番号4のデオキシリボヌクレオチド。
- 配列番号3である請求項1に記載のデオキシリボヌクレオチド。
- 配列番号4である請求項1に記載のデオキシリボヌクレオチド。
- 請求項1に記載のデオキシリボヌクレオチド及び薬学的に許容可能な担体を含む医薬組成物。
- 請求項4に記載の医薬組成物を含む近視を予防又は治療するための医薬組成物。
- 配列番号21の、ロックド核酸修飾及びホスホロチオエート結合修飾オリゴヌクレオチドであって、配列番号21の5’末端から3’末端まで1位、2位、3位、4位、17位、18位、19位及び20位はロックド核酸で修飾し、配列番号21の全てのホスホジエステル結合はホスホロチオエート結合で修飾することを特徴とするオリゴヌクレオチド。
- 請求項6に記載のロックド核酸修飾及びホスホロチオエート結合修飾オリゴヌクレオチドならびに薬学的に許容可能な担体を含む医薬組成物。
- 請求項6に記載の医薬組成物を含む近視を予防又は治療するための医薬組成物。
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