JP7253379B2 - 組織特異的発現のための改変u6プロモーターシステム - Google Patents
組織特異的発現のための改変u6プロモーターシステム Download PDFInfo
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- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Description
本出願は、開示の別個の部分として、コンピュータ可読形式の配列表(ファイル名:50393A_SeqListing.txt、1,684,397バイト、ASCIIテキストファイル)を含み、その全体は参照により本明細書に組み込まれる。
miRNA発現の過負荷は、骨格筋、肝臓、及び他の器官において潜在的に毒性である。したがって、遺伝子療法中のmiRNAの高発現の毒性を回避する手段としてmiRNA発現を指示する、より弱いプロモーターの開発が必要とされている。例えば、減弱化されたU6システムが、肝臓におけるC型肝炎ウイルス(HCV)についてのAAV8媒介性RNAi治療のために開発された。このシステムは現在、AAVを用いたRNAi治療の最初の臨床試験において試験されている。要約すると、この試験を裏付ける前臨床データは、野生型(WT)U6プロモーターによって産生されたshRNAがHCVを効果的に破壊したが、マウス及びサルにおいて肝細胞毒性も引き起こしたことを示した。WT U6プロモーターの重要な残基を変異させることは、HCV破壊の効力を維持しながら、U6転写を弱め、16倍少ないshRNAを生じることにより、これを軽減した(Suhy et al.,Mol.Ther.20:1737-49,2012,Safety and Efficacy Study of Single Doses of TT034 in Patients with Chronic Hepatitis C,clinicaltrial.gov,July 8,2013)。この例では、標的器官は肝臓であった。本発明は、肝臓を回避することを追い求めたものであり、したがって、遺伝子療法に使用され得る減弱化された骨格筋特異的U6プロモーターシステムを提供する。
(発明を実施するための形態)
本発明は、改変U6プロモーターシステムを提供し、これは引き続き標的遺伝子のmiRNA誘導阻害をもたらすか、あるいは標的遺伝子の阻害をもたらし得るmiRNAの置換をもたらすか、あるいは転写下にあるmiRNAの置換をもたらしてもよい。野生型U6プロモーター(U6-1)は、配列番号3として示される。一方、PSE領域内に置換を有する減弱化されたU6プロモーターは、図1に示されるように配列番号4として示される。
本発明のプロモーターシステムは、miRNAを非標的化するための結合部位が、miRNAの成熟ガイド鎖のループ内あるいはmiRNAの成熟ガイド鎖の5’または3’末端に挿入されている、miRNAの成熟ガイド鎖を含む核酸分子である。例えば、骨格筋におけるmiRNA配列の発現を促進し、肝臓及び心臓組織におけるmiRNAの発現を非標的化するために、核酸分子は、肝臓特異的miR-122の結合部位及び/または心臓特異的miR-208の結合部位が、成熟ガイド鎖のループ内またはmiRNAの成熟ガイド鎖の5’もしくは3’末端に挿入されている、miRNAの成熟ガイド鎖を含む。miRNA-122の結合部位のヌクレオチド配列は、配列番号5として示され、miRNA-208の結合部位のヌクレオチド配列は、配列番号6として示される。
本発明の核酸分子は、組織特異的発現を有することが所望される任意のmiRNA転写配列の成熟ガイド鎖の配列を含み得る。例えば、一実施形態では、DUX4 miRNAの骨格発現が企図される。例示的なDUX4 miRNA配列は、国際特許出願第PCT/US2012/047999号(WO 2013/016352)及び米国特許公開第US2012/20225034号に記載されており、その全体は参照により本明細書に組み込まれる。
本発明の組換えAAVゲノムは、本発明の核酸分子、及び核酸分子に隣接する1つ以上のAAV ITRを含む。rAAVゲノム中のAAV DNAは、任意のAAV血清型に由来してもよく、その組換えウイルスは、AAV血清型、AAV-B1、AAVrh.74、AAV-1、AAV-2、AAV-3、AAV-4、AAV-5、AAV-6、AAV-7、AAV-8、AAV-9、AAV-10、AAV-11、AAV-12、及びAAV-13を含むが、これらに限定されない。偽型rAAVの産生は、例えば、WO01/83692に開示されている。他の種類のrAAV変異体、例えば、カプシド変異を有するrAAVもまた企図される。例えば、Marsic et al.,Molecular Therapy,22(11):1900-1909(2014)を参照されたい。上記の背景技術の項で述べたように、種々のAAV血清型のゲノムのヌクレオチド配列は、当該技術分野で既知である。骨格筋特異的発現を促進するために、AAV1、AAV5、AAV6、AAV8、またはAAV9が使用されてもよい。
筋ジストロフィー(MD)は、遺伝性疾患群である。この群は、運動及び呼吸を制御する骨格筋の進行性の衰弱及び変性によって特徴付けられる。いくつかの形態のMDは幼年期または小児期に発症するが、中年以降まで発症しないものもある。この障害は、筋力の低下(いくつかの形態のMDは心筋にも影響を及ぼす)の分布及び程度、発病年齢、進行速度、ならびに遺伝パターンに関して異なる。
配列番号1(miDUX4.405またはmiDUX4-1)
肝臓及び心臓を非標的化した、減弱化されたU6プロモーターシステム
筋肉は、大過剰量のmiRNAベクターによる損傷を受けやすい。したがって、骨格筋特異的miRNA発現のための改変U6プロモーターシステムが開発された。野生型U6プロモーターを、図1に示されるような近位配列エレメントにおいて変異させた。この突然変異は、Suhy et al.,Mol.Therapy 20:1737-1749,2012に記載されているように、肝炎治療のためのHCV破壊の効力を維持しながら、U6転写を弱め、AAV8において16倍少ないshRNA転写を産生する。本実験では、ウミシイタケルシフェラーゼがDUX4配列を含むルシフェラーゼアッセイを用いて、miDUX4を駆動するこの減弱化されたU6(wU6)システムを含む核酸分子が、インビトロで有意なDUX4サイレンシングを達成した。(図3B)。
DUX4 miRNAの発現の効果のためのルシフェラーゼアッセイ
DUX4 miRNAの存在下で、DUX4標的配列の発現をアッセイした。リポフェクタミン2000トランスフェクションを、96ウェルの白壁アッセイプレート中の293細胞で行った。DUX4標的配列を含む20ngのウミシイタケ-ホタルのプラスミドと、実施例1のU6T6駆動miDux4.405またはmiDux4.1155ベクターを含む180ngの種々のDUX4 miRNAコード化ベクターとを用いて、140,000個の細胞をトランスフェクトした。24時間後にルシフェラーゼアッセイを実施した。
DUX4マイクロRNAをコードするrAAVの産生
HEK293細胞における3つのプラスミド[pAdhelper、AAVヘルパー、及びmiDUX4を含むrAAVゲノム、以下に詳細に記載される]の同時トランスフェクションによりrAAVベクターを産生し、次いで、細胞を回収し、ベクター精製、力価測定、及び品質管理アッセイを行った。
本発明は、例えば、以下の項目を提供する。
(項目1)
改変U6プロモーター配列、少なくとも1つの非標的配列を含むmiRNAの成熟ガイド鎖、及び5’末端に5~6つのチミジンを含む、核酸分子。
(項目2)
前記非標的配列が、miRNA-122またはmiRNA 208の結合部位である、項目1に記載の核酸分子。
(項目3)
前記改変U6プロモーター配列が、近位配列エレメント(PSE)領域または遠位配列エレメント(DSE)に少なくとも置換、挿入または欠失を含む、項目1または2に記載の核酸分子。
(項目4)
前記改変U6プロモーター配列が、PSE配列中のヌクレオチド-66のシトシンのチミジンへの置換、ヌクレオチド-57のシトシンのチミジンへの置換、及びヌクレオチド-52のチミジンのシトシンへの置換を含む、項目1または2に記載の核酸分子。
(項目5)
前記miRNAの成熟ガイド鎖が、miDUX4、miRN92、miRNA-17、miRNA-18a、miRNA-19a、miRNA-20a、miRNA-19b-1、mi-RNA-26a、miRNA-126、miRNA-335、let-7a 及びlet-7b、miRNA-34、miR-34a、miRNA-10b、miRNA-208、miRNA-499、miRNA-195、miRNA-29a、miRNA-29b、またはmiRNA-29cである、項目1~4のいずれか1項に記載の核酸分子。
(項目6)
前記miRNAの成熟ガイド鎖が、配列番号8482、配列番号8372、配列番号8371、配列番号8370、配列番号8367、配列番号8366、配列番号8365、配列番号8219、配列番号8218、配列番号8152、配列番号8147、配列番号8145、配列番号7397、配列番号7396、配列番号7395、配列番号7108、配列番号7107、配列番号7106、配列番号6633、配列番号6631、配列番号6622、配列番号6619、配列番号6609、配列番号6608、配列番号6568、配列番号6561、配列番号6560、配列番号10971、または配列番号10972のヌクレオチド配列を含む、項目1~4のいずれか1項に記載の核酸分子。
(項目7)
前記miRNAの成熟ガイド鎖がmiDUX4である、項目1~4のいずれか1項に記載の核酸分子。
(項目8)
配列番号1、2、または10913~10968のいずれか1つの核酸配列を含む、項目1~4のいずれか1項に記載の核酸分子。
(項目9)
項目1~8のいずれか1項に記載の核酸分子を含む、ベクター。
(項目10)
前記ベクターが、プラスミド、アデノ随伴ウイルス、アデノウイルス、レトロウイルス、レンチウイルス、ウマ随伴ウイルス、アルファウイルス、ポックスウイルス、ヘルペスウイルス、ポリオウイルス、シンドビスウイルス、またはワクシニアウイルスである、項目9に記載のベクター。
(項目11)
項目1~8のいずれか1項に記載の核酸分子を含む、組換えアデノ随伴ウイルス。
(項目12)
前記AAVが、AAV6、AAV rh.74、またはAAV-B1である、項目11に記載の組換えアデノ随伴ウイルス。
(項目13)
項目9もしくは10に記載のベクターまたは項目11もしくは12に記載の組換えアデノ随伴ウイルスを含む、組成物。
(項目14)
細胞内の遺伝子の発現を阻害する方法であって、項目1~8のいずれか1項に記載の核酸分子を含む組換えアデノ随伴ウイルスまたは項目13に記載の組成物と前記細胞を接触させることを含む、方法。
(項目15)
細胞におけるDUX4遺伝子の発現を阻害する方法であって、項目1~8のいずれか1項に記載の核酸分子を含む組換えアデノ随伴ウイルスまたは項目13に記載の組成物と前記細胞を接触させることを含む、方法。
(項目16)
DUX4 miRNAをコードするDNAをそれを必要とする動物の骨格筋に送達する方法であって、項目1~8のいずれか1項に記載の核酸分子を含む組換えアデノ随伴ウイルスまたは項目13に記載の組成物を前記動物に投与することを含む、方法。
(項目17)
顔面肩甲上腕型筋ジストロフィーを治療する方法であって、項目1~8のいずれか1項に記載の核酸分子を含む組換えアデノ随伴ウイルスまたは項目13に記載の組成物を投与することを含む、方法。
(項目18)
前記組換えアデノ随伴ウイルスが、筋肉内注射、経皮輸送、血流への注射、または肝臓への注射によって投与される、項目14~17のいずれか1項に記載の方法。
(項目19)
細胞内の遺伝子の発現を阻害するための医薬の調製のための、項目1~8のいずれか1項に記載の核酸分子を含む組換えアデノ随伴ウイルスまたは項目13に記載の組成物の使用。
(項目20)
細胞内のDUX4遺伝子の発現を阻害するための医薬の調製のための、項目1~8のいずれか1項に記載の核酸分子を含む組換えアデノ随伴ウイルスまたは項目13に記載の組成物の使用。
(項目21)
DUX4 miRNAをコードするDNAをそれを必要とする動物の骨格筋へ送達するための医薬の調製のための、項目1~8のいずれか1項に記載の核酸分子を含む組換えアデノ随伴ウイルスまたは項目13に記載の組成物の使用。
(項目22)
顔面肩甲上腕型筋ジストロフィーを治療するための医薬の調製のための、項目1~8のいずれか1項に記載の核酸分子を含む組換えアデノ随伴ウイルスまたは項目13に記載の組成物の使用。
(項目23)
前記医薬が、筋肉内注射、経皮輸送、または血流への注射のために製剤化される、項目19~22のいずれか1項に記載の使用。
(項目24)
細胞内の遺伝子の発現を阻害するための、項目1~8のいずれか1項に記載の核酸分子を含む組換えアデノ随伴ウイルスまたは項目13に記載の組成物を含む、組成物。
(項目25)
細胞内のDUX4遺伝子の発現を阻害するための、項目1~8のいずれか1項に記載の核酸分子を含む組換えアデノ随伴ウイルスまたは項目13に記載の組成物を含む、組成物。
(項目26)
DUX4 miRNAをコードするDNAをそれを必要とする動物の骨格筋へ送達するための、項目1~8のいずれか1項に記載の核酸分子を含む組換えアデノ随伴ウイルスまたは項目13に記載の組成物を含む、組成物。
(項目27)
顔面肩甲上腕型筋ジストロフィーを治療するための、項目1~8のいずれか1項に記載の核酸分子を含む組換えアデノ随伴ウイルスまたは項目13に記載の組成物を含む、組成物。
(項目28)
前記組成物が、筋肉内注射、経皮輸送、または血流への注射のために製剤化される、項目24~27のいずれか1項に記載の組成物。
Claims (22)
- (a)配列番号4のヌクレオチド配列を含む改変U6プロモーター配列、
(b)miRNA-122および/またはmiRNA-208の結合部位である、少なくとも1つの非標的配列を含むマイクロRNA(miRNA)の成熟ガイド鎖、及び
(c)5’末端に5~6つのチミジン
を含む、核酸。 - a)miRNA-122の結合部位が、配列番号5のヌクレオチド配列を含み、かつ/または
b)miRNA 208の結合部位が、配列番号6のヌクレオチド配列を含む、請求項1に記載の核酸。 - 前記miRNAの成熟ガイド鎖が、miDUX4、miRNA-92、miRNA-17、miRNA-18a、miRNA-19a、miRNA-20a、miRNA-19b-1、mi-RNA-26a、miRNA-126、miRNA-335、let-7a、let-7b、miRNA-34、miRNA-34a、miRNA-10b、miRNA-208、miRNA-499、miRNA-195、miRNA-29a、miRNA-29b、またはmiRNA-29cである、請求項1または2に記載の核酸。
- 前記miRNAの成熟ガイド鎖が、配列番号8482、配列番号8372、配列番号8371、配列番号8370、配列番号8367、配列番号8366、配列番号8365、配列番号8219、配列番号8218、配列番号8152、配列番号8147、配列番号8145、配列番号7397、配列番号7396、配列番号7395、配列番号7108、配列番号7107、配列番号7106、配列番号6633、配列番号6631、配列番号6622、配列番号6619、配列番号6609、配列番号6608、配列番号6568、配列番号6561、配列番号6560、配列番号10971、または配列番号10972のヌクレオチド配列を含む、請求項1または2に記載の核酸。
- 前記miRNAの成熟ガイド鎖がmiDUX4である、請求項1または2に記載の核酸。
- 配列番号1、2、または10913~10968のいずれか1つのヌクレオチド配列を含む、請求項1または2に記載の核酸。
- 請求項1~6のいずれか1項に記載の核酸を含む、ベクター。
- 前記ベクターが、プラスミド、アデノ随伴ウイルス、アデノウイルス、レトロウイルス、レンチウイルス、ウマ随伴ウイルス、アルファウイルス、ポックスウイルス、ヘルペスウイルス、ポリオウイルス、シンドビスウイルス、またはワクシニアウイルスである、請求項7に記載のベクター。
- 請求項1~6のいずれか1項に記載の核酸を含む、組換えアデノ随伴ウイルス(AAV)。
- 前記AAVが、AAV6、AAV rh.74、またはAAV-B1である、請求項9に記載の組換えアデノ随伴ウイルス。
- 請求項7もしくは8に記載のベクターまたは請求項9もしくは10に記載の組換えアデノ随伴ウイルスを含む、組成物。
- 細胞内の遺伝子の発現を阻害するための医薬の調製のための、請求項1~6のいずれか1項に記載の核酸を含む組換えアデノ随伴ウイルスまたは請求項11に記載の組成物の使用。
- 細胞内のDUX4遺伝子の発現を阻害するための医薬の調製のための、請求項1~6のいずれか1項に記載の核酸を含む組換えアデノ随伴ウイルスまたは請求項11に記載の組成物の使用。
- DUX4 miRNAをコードするDNAをそれを必要とする動物の骨格筋へ送達するための医薬の調製のための、請求項1~6のいずれか1項に記載の核酸を含む組換えアデノ随伴ウイルスまたは請求項11に記載の組成物の使用。
- 顔面肩甲上腕型筋ジストロフィーを治療するための医薬の調製のための、請求項1~6のいずれか1項に記載の核酸を含む組換えアデノ随伴ウイルスまたは請求項11に記載の組成物の使用。
- 前記医薬が、筋肉内注射、経皮輸送、または血流への注射のために製剤化される、請求項12~15のいずれか1項に記載の使用。
- 細胞内の遺伝子の発現を阻害するための、請求項1~6のいずれか1項に記載の核酸を含む組換えアデノ随伴ウイルスまたは請求項11に記載の組成物を含む、組成物。
- 細胞内のDUX4遺伝子の発現を阻害するための、請求項1~6のいずれか1項に記載の核酸を含む組換えアデノ随伴ウイルスまたは請求項11に記載の組成物を含む、組成物。
- DUX4 miRNAをコードするDNAをそれを必要とする動物の骨格筋へ送達するための、請求項1~6のいずれか1項に記載の核酸を含む組換えアデノ随伴ウイルスまたは請求項11に記載の組成物を含む、組成物。
- 顔面肩甲上腕型筋ジストロフィーを治療するための、請求項1~6のいずれか1項に記載の核酸を含む組換えアデノ随伴ウイルスまたは請求項11に記載の組成物を含む、組成物。
- 前記組成物が、筋肉内注射、経皮輸送、または血流への注射のために製剤化される、請求項17~20のいずれか1項に記載の組成物。
- 前記組成物が、筋肉内注射、経皮輸送、血流への注射または肝臓への注射によって投与される、請求項17~20のいずれか一項の記載の組成物。
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MX2023002853A (es) | 2020-09-11 | 2023-03-31 | Arrowhead Pharmaceuticals Inc | Agentes de arni para inhibir la expresion de dux4, composiciones de dichos agentes, y metodos de uso. |
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US20190136235A1 (en) | 2019-05-09 |
AU2017240236A1 (en) | 2018-10-18 |
KR102453187B1 (ko) | 2022-10-07 |
BR112018070249A2 (pt) | 2019-01-29 |
AU2017240236B2 (en) | 2023-07-27 |
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CA3019832C (en) | 2023-05-09 |
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US11939579B2 (en) | 2024-03-26 |
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