JP7007273B2 - 遺伝子治療用の改良された複合型二重組換えaavベクターシステム - Google Patents
遺伝子治療用の改良された複合型二重組換えaavベクターシステム Download PDFInfo
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Description
(a)以下を含む第1の核酸配列:
- スプライシングドナー(SD)シグナルの核酸配列(配列番号1)を含む合成イントロンの核酸配列の5'末端部分、及び
- 組換え誘導(recombinogenic)領域の核酸配列、並びに、
(b)以下を含む第2の核酸配列:
- 組換え誘導領域の核酸配列、及び
- 分岐部位及びポリピリミジントラクト及びスプライシングアクセプター(SA)シグナル(配列番号2)の核酸配列を含む合成イントロンの核酸配列の3'末端部分。
本明細書の全体にわたり、幾つかの用語が使用され、また以下の段落の通り定義される。
第1の態様において、本発明は、以下の一対の核酸配列から構成される二重構築物に関する:
(a)以下を含む第1の核酸配列:
- スプライシングドナー(SD)シグナルの核酸配列(配列番号1)を含む合成イントロンの核酸配列の5'末端部分、及び
- 組換え誘導領域の核酸配列、並びに、
(b)以下を含む第2の核酸配列:
- 組換え誘導領域の核酸配列、及び
- 分岐部位及びポリピリミジントラクト及びスプライシングアクセプター(SA)シグナルの核酸配列(配列番号2)を含む合成イントロンの核酸配列の3'末端部分。
第2の態様において、本発明は、宿主細胞における目的遺伝子のコーディング配列の発現に適する、以下を含む複合型二重構築物システムに関する:
a)5'-3'の方向に向かって、以下を含む第1のポリヌクレオチド:
- 5'逆向き末端反復(5'-ITR)配列、
- プロモーター配列、
- 前記コーディング配列の5'末端部分であって、前記プロモーターに作動可能に連結されその制御下にある5'末端部、
- スプライシングドナー(SD)シグナルの核酸配列(配列番号1)を含む合成イントロンの配列の5'末端部分、
- 組換え誘導領域の核酸配列、及び
- 3'逆向き末端反復(3'-ITR)配列、
並びに、
b) 5'-3'の方向に向かって、以下を含む第2のポリヌクレオチド:
- 5'逆向き末端反復(5'-ITR)配列、
- 組換え誘導領域の核酸配列、
- 分岐部位、ポリピリミジントラクト及びスプライシングアクセプター(SA)シグナル(配列番号2)を含む合成イントロンの配列の3'末端部分、
- 前記コーディング配列の3'末端、
- ポリアデニル化(pA)シグナル核酸配列、及び
- 3'逆向き末端反復(3'-ITR)配列。
第3の態様において、本発明は、宿主細胞における目的遺伝子のコーディング配列の発現に適する、以下を含む複合型二重rAAV(hdrAAV)ベクターシステムに関する:
a)5'-3'の方向に向かって、以下を含む第1のポリヌクレオチドを含む第1のrAAVベクター:
- 5'逆向き末端反復(5'-ITR)配列、
- プロモーター配列、
- 前記コーディング配列の5'末端部分であって、前記プロモーターに作動可能に連結されその制御下にある5'末端部、
- スプライシングドナー(SD)シグナルの核酸配列(配列番号1)を含む合成イントロンの配列の5'末端部分、
- 組換え誘導領域の核酸配列、及び
- 3'逆向き末端反復(3'-ITR)配列、
並びに、
b)5'-3'の方向に向かって、以下を含む第2のポリヌクレオチドを含む第2のrAAVベクター:
- 5'逆向き末端反復(5'-ITR)配列、
- 組換え誘導領域の核酸配列、
- 分岐部位及びポリピリミジントラクトを含む合成イントロンの配列の3'末端部分(配列番号2)、
- スプライシングアクセプター(SA)シグナルの核酸配列、
- 前記コーディング配列の3'末端、
- ポリアデニル化(pA)シグナル核酸配列、及び
- 3'逆向き末端反復(3'-ITR)配列。
本明細書における「宿主細胞又は遺伝子組換え宿主細胞」という用語は、前記構築物によって、又は前記ベクターを、形質導入され、形質転換され、又はトランスフェクションされた宿主細胞を関する。適切な宿主細胞の代表例として、細菌細胞(例えば大腸菌、ストレプトマイセス属菌、チフス菌(Salmonella typhimurium)、酵母等の菌類の細胞、Sf9等の昆虫細胞、動物細胞(例えばCHO又はCOS)、植物細胞等が挙げられる。適切な宿主の選択は、本明細書の教示から、当業者の技量の範囲内であると考えられる。好ましくは、前記宿主細胞は動物細胞、最も好ましくはヒト細胞である。本発明は更に、本明細書に記載されるいずれかの組換え発現ベクターを含む宿主細胞を提供する。前記宿主細胞は培養細胞又は初代細胞、すなわち生物(例えばヒト)から直接に単離された細胞でありうる。前記宿主細胞は、付着型の細胞又は懸濁型の細胞(すなわち懸濁液中で増殖する細胞)でありうる。適切な宿主細胞は、従来技術において、公知であり、例えば、大腸菌DH5α細胞、チャイニーズハムスター卵細胞(CHO)、サルVERO細胞及びCOS細胞、ヒトHEK293及びHeLa細胞等が挙げられる。
本発明の第4の態様は、本発明による前記複合型二重構築物システム、本発明による前記複合型二重ウイルスベクターシステム、又は本発明による前記宿主細胞と、薬理学的に許容されるビヒクルとを含む医薬組成物に関する。
本発明の第5の態様は、薬物としての、好ましくは遺伝子治療における使用のための、更に好ましくは網膜変性を特徴とする病理又は疾患の治療及び/又は予防方法における使用のための、本発明の複合型二重構築物システム、本発明の複合型二重ウイルスベクターシステム又は本発明の宿主細胞に関する。
プラスミドの構築:スプリットABCA4のコーディング配列(Genbank NM_000350.2)、イントロン配列、APから派生した組換え誘導配列及び短いポリアデニル化配列を、公表された配列に基づきin silicoにおいて、設計した。設計された配列を含むDNAを、遺伝子合成によって、得た。
二重AAV2/5-ABCA4ベクターの効果的な産生:本発明の二重ベクター、AAV2/5-CMV-5'ABCA4-mAP及びAAV2/5-mAP3'ABCA4の産生は、前記ベクターゲノムのかなりのサイズにもかかわらず効果的であり、それは、AAVの最大のパッケージング能力付近、すなわち約5.1~5.2kbであった(Wuら、Mol Ther 2010)。データ(Table 1(表1))から、適正な産生レベル(CellStack-5培養チャンバー当たり約1013vg)を示し、全体(vg)に対する感染性粒子の比率(ベクターの品質の指標)は、標準的な正規のサイズのAAV2/5ベクターのそれに等しかった。
Abca4-/-マウスの目への、1.4×109vgのAAV2/5-CMV-5'ABCA4-mAP又は1.5~5×109vgのAAV2/5-RK-5'ABCA4-mAPベクター、及び1.2×109vg又は5×109vgのAAV2/5-mAP-3'ABCA4の網膜下投与後、逆転写された神経網膜RNAの定量的PCR解析を上記の通りに実施した(図10)。前記二重ベクターにより注射されたAbca4-/-マウスにおいてのみ、ヒトのABCA4エクソン21-エクソン22接合部に対応する183bp増幅産物が検出され、注射を受けていない野生型(Abca4+/+)及びAbca4-/-マウスでは検出されなかった。注射された神経網膜におけるヒトABCA4mRNAの発現レベルは、前記光受容体に特異的なRKプロモーターから発現させたとき、ユビキタスCMVプロモーターと比較し、用量に関係なく、約1.6倍高かった
Claims (16)
- 宿主細胞における目的遺伝子のコーディング配列の発現に適する複合型二重構築物システムであって、以下:
a)5'-3'の方向に向かって、以下を含む第1のポリヌクレオチド:
- 5'逆向き末端反復(5'-ITR)配列、
- ヒトロドプシンキナーゼ(RK)のプロモーター配列、
- 前記コーディング配列の5'末端部分であって、前記プロモーターに作動可能に連結され前記プロモーターの制御下にある、5'末端部分、
- スプライシングドナー(SD)シグナルの核酸配列(配列番号1)を含む合成イントロンの配列の5'末端部分、
- アルカリホスファターゼ(AP)から派生したか又はこれに由来する組換え誘導(recombinogenic)領域の核酸配列、並びに
- 3'逆向き末端反復(3'-ITR)配列、
並びに
b)5'-3'の方向に向かって、以下を含む第2のポリヌクレオチド:
- 5'逆向き末端反復(5'-ITR)配列、
- アルカリホスファターゼ(AP)から派生したか又はこれに由来する組換え誘導領域の核酸配列、
- 分岐部位、ポリピリミジントラクト及びスプライシングアクセプター(SA)シグナル(配列番号2)を含む合成イントロンの配列の3'末端部分、
- 前記コーディング配列の3'末端、
- ポリアデニル化(pA)シグナル核酸配列、並びに
- 3'逆向き末端反復(3'-ITR)配列
を含み、
前記組換え誘導領域のAPが、配列番号3の配列、又は配列番号6の派生コドン改変(mAP)配列を有し、前記コーディング配列が、ABCA4遺伝子である、
複合型二重構築物システム。 - ITRのヌクレオチド配列が、同じAAV血清型から、又は異なるAAV血清型から派生したものである、請求項1に記載の複合型二重構築物システム。
- 第1のプラスミドの3'-ITR及び第2のプラスミドの5'-ITRが、同じAAV血清型に由来する、請求項1又は2に記載の複合型二重構築物システム。
- 第1のプラスミドの5'-ITR及び3'-ITR、並びに第2のプラスミドの5'-ITR及び3'-ITRが、それぞれ異なるAAV血清型に由来する、請求項1又は2に記載の複合型二重構築物システム。
- 第1のプラスミドの5'-ITR、及び第2のプラスミドの3'-ITRが、異なるAAV血清型に由来する、請求項1又は2に記載の複合型二重構築物システム。
- 前記コーディング配列が、遺伝性の網膜変性を修正することが可能なタンパク質をコードするヌクレオチド配列である、請求項1から5のいずれか一項に記載の複合型二重構築物システム。
- 前記コーディング配列が、遺伝病を修正することが可能なタンパク質をコードするヌクレオチド配列である、請求項1から5のいずれか一項に記載の複合型二重構築物システム。
- 前記コーディング配列の5'末端部分が、配列番号7(エクソン1~21)の配列を有し、前記コーディング配列の3'末端が、配列番号8(エクソン22~50)の配列を有する、請求項1に記載の複合型二重構築物システム。
- 前記第1のポリヌクレオチドが、配列番号10(RK-5'ABCA4-SD-AP)の配列を含み、前記第2のポリヌクレオチドが、配列番号11(AP-SA-3'ABCA4-pA)の配列を含む、請求項1に記載の複合型二重構築物システム。
- 宿主細胞における目的遺伝子のコーディング配列の発現に適する複合型二重rAAV(hdrAAV)ベクターシステムであって、以下:
a)5'-3'の方向に向かって、以下を含む第1のポリヌクレオチドを含む第1のrAAVベクター:
- 5'逆向き末端反復(5'-ITR)配列、
- ヒトロドプシンキナーゼ(RK)のプロモーター配列、
- 前記コーディング配列の5'末端部分であって、前記プロモーターに作動可能に連結され前記プロモーターの制御下にある、5'末端部分、
- スプライシングドナー(SD)シグナルの核酸配列(配列番号1)を含む合成イントロンの配列の5'末端部分、
- アルカリホスファターゼ(AP)から派生したか又はこれに由来する組換え誘導(recombinogenic)領域の核酸配列、並びに
- 3'逆向き末端反復(3'-ITR)配列、
並びに
b)5'-3'の方向に向かって、以下を含む第2のポリヌクレオチドを含む第2のrAAVベクター:
- 5'逆向き末端反復(5'-ITR)配列、
- アルカリホスファターゼ(AP)から派生したか又はこれに由来する組換え誘導領域の核酸配列、
- 分岐部位及びポリピリミジントラクト及びスプライシングアクセプター(SA)シグナルの核酸配列(配列番号2)を含む合成イントロンの配列の3'末端部分、
- 前記コーディング配列の3'末端、
- ポリアデニル化シグナル核酸配列、並びに
- 3'逆向き末端反復(3'-ITR)配列
を含み、
前記組換え誘導領域のAPが、配列番号3の配列、又は配列番号6の派生コドン改変(mAP)配列を有し、前記コーディング配列が、ABCA4遺伝子である、
複合型二重rAAV(hdrAAV)ベクターシステム。 - 前記組換えAAVベクターが、血清型2、血清型4、血清型5及び血清型8から選択される、請求項10に記載のhdrAAVベクターシステム。
- 前記第1のrAAVベクターが、配列番号13(AAV-RK-5'ABCA4-mAPベクター)の配列を含み、前記第2のrAAVベクターが、配列番号14(AAV-mAP-SA-3'ABCA4-pAベクター)の配列を含む、請求項10に記載のhdrAAVベクターシステム。
- 請求項1から9のいずれか一項に記載の複合型二重構築物システム又は請求項10から12のいずれか一項に記載のhdrAAVベクターシステムを、形質導入、形質転換又はトランスフェクションされた宿主細胞。
- 請求項10から12のいずれか一項に記載のhdrAAVベクターシステム又は請求項13に記載の宿主細胞と、薬理学的に許容されるビヒクルとを含む医薬組成物。
- 薬物としての使用のための、請求項10から12のいずれか一項に記載のhdrAAVベクターシステム又は請求項13に記載の宿主細胞を含む医薬組成物。
- それを必要とする被験体における網膜変性を特徴とする病理又は疾患の治療及び/又は予防のための、請求項15に記載の医薬組成物。
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