JP2022514761A - ウイルスベクターの自動産生方法 - Google Patents
ウイルスベクターの自動産生方法 Download PDFInfo
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Abstract
Description
一部の実施形態では、本明細書において、アデノ随伴ウイルス(AAV)ウイルスベクターの自動産生方法が提供され、当該方法は、人工哺乳類AAVウイルス産生細胞を完全密閉式細胞工学システムに導入することであって、人工哺乳類AAVウイルス産生細胞が、そのゲノムに組み込まれた、第1の抑制解除プロモータの制御下のE2AおよびE4Orf6遺伝子を含むアデノウイルスヘルパー遺伝子、第2の抑制解除プロモータの制御下のRepおよびCap遺伝子を含むAAV遺伝子、第3の抑制解除プロモータの制御下のウイルス関連非コードRNA、ならびに第1、第2、および第3の抑制解除プロモータの抑制要素を含む、導入することと、対象遺伝子をコードするベクターで哺乳類AAVウイルス産生細胞を形質導入して、形質導入ウイルス産生細胞を産生することと、抑制要素の結合パートナーで哺乳類AAVウイルス産生細胞を治療することと、第1、第2、および第3の抑制解除プロモータを活性化することと、形質導入ウイルス産生細胞を増殖させ、形質導入ウイルス産生細胞内でAAVウイルスベクターを産生することと、ウイルスベクターを単離することと、を含み、上記ステップは、閉鎖型自動化プロセスで行われる。
実施形態1は、アデノ随伴ウイルス(AAV)ウイルスベクターの自動産生方法であって、当該方法は、人工哺乳類AAVウイルス産生細胞を完全密閉式細胞工学システムに導入することであって、人工哺乳類AAVウイルス産生細胞が、そのゲノムに組み込まれた、第1の抑制解除プロモータの制御下のE2AおよびE4Orf6遺伝子を含むアデノウイルスヘルパー遺伝子、第2の抑制解除プロモータの制御下のRepおよびCap遺伝子を含むAAV遺伝子、第3の抑制解除プロモータの制御下のウイルス関連非コードRNA、ならびに第1、第2、および第3の抑制解除プロモータの抑制要素を含む、導入することと、対象遺伝子をコードするベクターで哺乳類AAVウイルス産生細胞を形質導入して、形質導入ウイルス産生細胞を産生することと、抑制要素の結合パートナーで哺乳類AAVウイルス産生細胞を治療することと、第1、第2、および第3の抑制解除プロモータを活性化することと、形質導入ウイルス産生細胞を増殖させ、形質導入ウイルス産生細胞内でAAVウイルスベクターを産生することと、ウイルスベクターを単離すること、を含み、上記ステップは、閉鎖型自動化プロセスで行われる。
Claims (56)
- アデノ随伴ウイルス(AAV)ウイルスベクターの自動産生方法であって、
(a)人工哺乳類AAVウイルス産生細胞を完全密閉式細胞工学システムに導入することであって、前記人工哺乳類AAVウイルス産生細胞が、そのゲノムに組み込まれた、
i.第1の抑制解除プロモータの制御下のE2AおよびE4Orf6遺伝子を含むアデノウイルスヘルパー遺伝子、
ii.第2の抑制解除プロモータの制御下のRepおよびCap遺伝子を含むAAV遺伝子、
iii.第3の抑制解除プロモータの制御下のウイルス関連非コードRNA、ならびに
iv.前記第1、第2、および第3の抑制解除プロモータの抑制要素、を含む、導入することと、
(b)対象遺伝子をコードするベクターで前記哺乳類AAVウイルス産生細胞を形質導入して、形質導入ウイルス産生細胞を産生することと、
(c)前記抑制要素の結合パートナーで前記哺乳類AAVウイルス産生細胞を治療することと、
(d)前記第1、第2、および第3の抑制解除プロモータを活性化することと、
(e)前記形質導入ウイルス産生細胞を増殖させ、前記形質導入ウイルス産生細胞内で前記AAVウイルスベクターを産生することと、
(f)前記ウイルスベクターを単離することと、を含み、
(a)~(f)が、閉鎖型自動化プロセスで行われる、方法。 - 前記人工哺乳類AAVウイルス産生細胞が、哺乳類細胞培養物である、請求項1に記載の方法。
- 前記哺乳類細胞培養物が、懸濁培養物である、請求項2に記載の方法。
- 前記人工哺乳類AAVウイルス産生細胞が、チャイニーズハムスター卵巣(CHO)細胞である、請求項1~3のいずれか一項に記載の方法。
- 前記人工哺乳類AAVウイルス産生細胞が、ヒト細胞である、請求項1~3のいずれか一項に記載の方法。
- 前記ヒト細胞が、ヒト胚腎臓(HEK)細胞である、請求項5に記載の方法。
- 前記抑制解除プロモータの各々が、機能的プロモータおよび2つのテトラサイクリンオペレータ配列(TetO2 )を含み、前記抑制要素が、テトラサイクリン抑制タンパク質である、請求項1~6のいずれか一項に記載の方法。
- 前記治療することが、ドキシサイクリンで治療することを含む、請求項7に記載の方法。
- 前記対象遺伝子が、治療対象遺伝子である、請求項1~8のいずれか一項に記載の方法。
- 産生されたAAVウイルスベクターの量が、少なくとも約1010個のウイルスベクターである、請求項1~9のいずれか一項に記載の方法。
- 前記閉鎖型自動化プロセスが、
(a)温度センサ、pHセンサ、グルコースセンサ、ラクトースセンサ、酸素センサ、二酸化炭素センサ、および光学密度センサのうちの1つ以上で監視することと、
(b)温度、pHレベル、グルコースレベル、ラクトースレベル、酸素レベル、二酸化炭素レベル、および光学密度のうちの1つ以上を自動的に調節することと、を含む、請求項1~10のいずれか一項に記載の方法。 - 前記形質導入することが、ウイルス感染、エレクトロポレーション、リポソームトランスフェクション、または膜破壊を含む、請求項1~11のいずれか一項に記載の方法。
- アデノ随伴ウイルス(AAV)ウイルスベクターの自動産生方法であって、
(a)TetRおよび/またはTetR-KRABをコードする1つ以上の核酸を安定的に発現する哺乳類細胞を、
i.第1の抑制解除プロモータの制御下でE2A遺伝子、E4Orf遺伝子、およびウイルス関連非コードRNAを含むアデノウイルスヘルパー遺伝子をコードする第1の核酸、
ii.第2の抑制解除プロモータの制御下でRepおよびCap遺伝子を含むAAV遺伝子をコードする第2の核酸、および
iii.任意選択で、第3の抑制解除プロモータの制御下で対象遺伝子をコードする第3の核酸によって形質導入することと、
(b)前記哺乳類細胞を前記TetRの結合パートナーで治療することと、
(c)前記第1、第2、および第3の抑制解除プロモータを活性化することと、
(d)前記形質導入細胞を増殖させ、前記形質導入細胞内で前記AAVウイルスベクターを産生することと、
(e)前記ウイルスベクターを単離することと、を含み、
(a)~(e)が、閉鎖型自動化プロセスで行われる、方法。 - 前記哺乳類細胞が、哺乳類細胞培養物である、請求項13に記載の方法。
- 前記哺乳類細胞培養物が、懸濁培養物である、請求項14に記載の方法。
- 前記哺乳類細胞が、チャイニーズハムスター卵巣(CHO)細胞である、請求項12~15のいずれか一項に記載の方法。
- 前記哺乳類細胞が、ヒト細胞である、請求項12~15のいずれか一項に記載の方法。
- 前記ヒト細胞が、ヒト胚腎臓(HEK)細胞である、請求項17に記載の方法。
- 前記治療することが、ドキシサイクリンで治療することを含む、請求項12~18のいずれか一項に記載の方法。
- 前記対象遺伝子が、治療対象遺伝子である、請求項12~19のいずれか一項に記載の方法。
- 産生されたAAVウイルスベクターの量が、少なくとも約1010個のウイルスベクターである、請求項12~20のいずれか一項に記載の方法。
- 前記閉鎖型自動化プロセスが、
(a)温度センサ、pHセンサ、グルコースセンサ、ラクトースセンサ、酸素センサ、二酸化炭素センサ、および光学密度センサのうちの1つ以上で監視することと、
(b)温度、pHレベル、グルコースレベル、ラクトースレベル、酸素レベル、二酸化炭素レベル、および光学密度のうちの1つ以上を自動的に調節することと、を含む、請求項12~21のいずれか一項に記載の方法。 - 前記形質導入することが、ウイルス感染、エレクトロポレーション、リポソームトランスフェクション、または膜破壊を含む、請求項12~22のいずれか一項に記載の方法。
- レンチウイルスベクターの自動産生方法であって、
(a)人工哺乳類レンチウイルスベクター産生細胞を完全密閉式細胞工学システムに導入することであって、前記人工哺乳類レンチウイルスベクター産生細胞が、そのゲノムに組み込まれた、
i.第1のプロモータの制御下のビリオンタンパク質(REV)遺伝子の発現のレンチウイルス調節因子、
ii.第2のプロモータの制御下のレンチウイルスエンベロープ遺伝子、ならびに
iii.ともに第3のプロモータの制御下のレンチウイルス群特異抗原(GAG)遺伝子およびレンチウイルスポリメラーゼ(POL)遺伝子、を含み、
核酸配列が、トランスポゾン特異的逆方向末端反復(ITR)の組換えから生じる配列によって5’末端および3’末端の両方に隣接されている、導入することと、
(b)対象遺伝子をコードするベクターで前記哺乳類レンチウイルスベクター産生細胞を形質導入して、形質導入ウイルス産生細胞を産生することと、
(c)前記第1、第2、および第3のプロモータを活性化することと、
(d)前記形質導入ウイルス産生細胞を増殖させ、前記形質導入ウイルス産生細胞内で前記レンチウイルスベクターを産生することと、
(e)前記ウイルスベクターを単離することと、を含み、
(a)~(e)が、閉鎖型自動化プロセスで行われる、方法。 - 前記人工哺乳類レンチウイルスベクター産生細胞が、哺乳類細胞培養物である、請求項24に記載の方法。
- 前記哺乳類細胞培養物が、懸濁培養物である、請求項25に記載の方法。
- 前記人工哺乳類レンチウイルスベクター産生細胞が、ヒト細胞である、請求項24~26のいずれか一項に記載の方法。
- 前記ヒト細胞が、ヒト胚腎臓(HEK)細胞である、請求項27に記載の方法。
- 前記GAG遺伝子が、HIV GAG遺伝子であり、前記POL遺伝子が、HIV POL遺伝子である、請求項24~28のいずれか一項に記載の方法。
- 前記レンチウイルスエンベロープ遺伝子が、水疱口炎ウイルス糖タンパク質(VSV-G)遺伝子である、請求項24~29のいずれか一項に記載の方法。
- 前記第1のプロモータ、第2のプロモータ、および第3のプロモータが、抑制解除プロモータである、請求項24~30のいずれか一項に記載の方法。
- 前記人工哺乳類レンチウイルスベクター産生細胞が、前記第1、第2、および第3の抑制解除プロモータの抑制要素をそのゲノムに組み込むことをさらに含む、請求項31に記載の方法。
- 前記抑制解除プロモータの各々が、機能的プロモータおよびテトラサイクリンオペレータ配列(TetO)を含み、前記抑制要素が、テトラサイクリン抑制タンパク質である、請求項32に記載の方法。
- 前記テトラサイクリン抑制タンパク質をコードする配列に続く、クルッペル会合ボックス配列をさらに含む、請求項33に記載の方法。
- 前記トランスポゾン特異的ITRが、レピドプテラントランスポゾン(PIGGYBAC (登録商標)) ITRである、請求項24~34のいずれか一項に記載の方法。
- 前記対象遺伝子が、治療対象遺伝子である、請求項24~35のいずれか一項に記載の方法。
- 産生されたレンチウイルスベクターの量が、少なくとも約1010個のウイルスベクターである、請求項24~36のいずれか一項に記載の方法。
- 前記閉鎖型自動化プロセスが、
(a)温度センサ、pHセンサ、グルコースセンサ、ラクトースセンサ、酸素センサ、二酸化炭素センサ、および光学密度センサのうちの1つ以上で監視することと、
(b)温度、pHレベル、グルコースレベル、ラクトースレベル、酸素レベル、二酸化炭素レベル、および光学密度のうちの1つ以上を自動的に調節することと、を含む、請求項24~37のいずれか一項に記載の方法。 - 前記形質導入することが、ウイルス感染、エレクトロポレーション、リポソームトランスフェクション、または膜破壊を含む、請求項24~38のいずれか一項に記載の方法。
- レンチウイルスベクターの自動産生方法であって、
(a)完全密閉式細胞工学システムに哺乳類細胞を導入することと、
(b)哺乳類細胞を、
i.第1のプロモータの制御下のビリオンタンパク質(REV)遺伝子の発現のレンチウイルス調節因子ならびに第2のプロモータの制御下のエンベロープ糖タンパク質遺伝子をコードする第1の核酸、
ii.第3のプロモータの制御下の対象遺伝子をコードする第2の核酸、および
iii.第4のプロモータの制御下のレンチウイルス群特異抗原(GAG)遺伝子ならびにレンチウイルスポリメラーゼ(POL)遺伝子の両方をコードする第3の核酸によって形質導入することと、
(c)前記形質導入細胞を増殖させ、前記形質導入細胞内で前記レンチウイルスベクターを産生することと、
(d)前記ウイルスベクターを単離することと、を含み、
(a)~(d)が、閉鎖型自動化プロセスで行われる、方法。 - 前記哺乳類細胞が、哺乳類細胞培養物である、請求項40に記載の方法。
- 前記哺乳類細胞培養物が、懸濁培養物である、請求項41に記載の方法。
- 前記哺乳類細胞が、ヒト細胞である、請求項40~42のいずれか一項に記載の方法。
- 前記ヒト細胞が、ヒト胚腎臓(HEK)細胞である、請求項43に記載の方法。
- 前記GAG遺伝子が、HIV GAG遺伝子であり、前記POL遺伝子が、HIV POL遺伝子である、請求項40~44のいずれか一項に記載の方法。
- 前記エンベロープ糖タンパク質遺伝子が、水疱口炎ウイルス糖タンパク質(VSV-G)遺伝子であり、前記VSV-G遺伝子および前記REV遺伝子が、コドン最適化される、請求項40~46のいずれか一項に記載の方法。
- 前記対象遺伝子が、治療対象遺伝子である、請求項40~46のいずれか一項に記載の方法。
- 産生されたレンチウイルスベクターの量が、少なくとも約1010個のウイルスベクターである、請求項40~47のいずれか一項に記載の方法。
- 前記閉鎖型自動化プロセスが、
(a)温度センサ、pHセンサ、グルコースセンサ、ラクトースセンサ、酸素センサ、二酸化炭素センサ、および光学密度センサのうちの1つ以上で監視することと、
(b)温度、pHレベル、グルコースレベル、ラクトースレベル、酸素レベル、二酸化炭素レベル、および光学密度のうちの1つ以上を自動的に調節することと、を含む、請求項40~48のいずれか一項に記載の方法。 - 前記形質導入することが、ウイルス感染、エレクトロポレーション、リポソームトランスフェクション、または膜破壊を含む、請求項40~49のいずれか一項に記載の方法。
- 請求項1~23のいずれか一項に記載の方法によって産生されるAAVウイルスベクター。
- 請求項14~50のいずれか一項に記載の方法によって産生されるレンチウイルスベクター。
- AAVベクターによる治療方法であって、
a.哺乳類対象に請求項51に記載のAAVベクターを投与することを含む、方法。 - 前記投与することが、吸入、注射、または静脈内投与を含む、請求項53に記載の方法。
- レンチウイルスベクターによる治療方法であって、
a.哺乳類対象に請求項52に記載のレンチウイルスベクターを投与することを含む、方法。 - 前記投与することが、吸入、注射、または静脈内投与を含む、請求項55に記載の方法。
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CN113195725A (zh) | 2021-07-30 |
CA3123449A1 (en) | 2020-06-25 |
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