JP7161730B2 - 顆粒状角膜変性症に対する遺伝子治療薬 - Google Patents
顆粒状角膜変性症に対する遺伝子治療薬 Download PDFInfo
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Description
(1)対象における顆粒状角膜変性症を治療する方法であって、
顆粒状角膜変性症に関連するトランスフォーミング増殖因子β誘発(TGFBI)遺伝子の変異を修復するために、対象の角膜実質細胞とCas9及びガイドRNAとを接触させる工程を含む、方法。
(2)TGFBI遺伝子の変異が点変異である、(1)に記載の方法。
(3)顆粒状角膜変性症が顆粒状角膜ジストロフィー2型(GCD2)である、(1)又は(2)に記載の方法。
(4)点変異がTGFBIタンパク質の124位におけるアミノ酸置換である、(2)に記載の方法。
(5)アミノ酸置換がR124H、R124C及びR124Lからなる群から選択される、(4)に記載の方法。
(6)ガイドRNAが、TGFBI遺伝子のスペーサー前隣接モチーフ(PAM)配列の上流側に隣接した22塩基の配列から成る標的配列とハイブリダイズすることができるガイド配列を含む、(1)~(5)のいずれかに記載の方法。
(7)PAM配列がCGGから成る、(6)に記載の方法。
(8)ガイド配列が、標的配列と相補的な、少なくとも17~18塩基から成る領域を含む、(6)又は(7)に記載の方法。
(9)ガイド配列が、アデニン又はグアニンから始まる22塩基の配列から成る、(6)~(8)のいずれかに記載の方法。
(10)ガイド配列が配列番号1の塩基配列を有する、(9)に記載の方法。
(11)ガイド配列が、trans-activating crRNA(tracr RNA)配列と結合している、(6)~(10)のいずれかに記載の方法。
(12)ガイドRNAの発現がU6プロモーターによって駆動される、(1)~(11)のいずれかに記載の方法。
(13)ガイドRNAとU6プロモーターが同一又は異なるベクター上に位置する、(12)に記載の方法。
(14)ガイドRNAとU6プロモーターが同一のベクター上で作用可能に連結している、(13)に記載の方法。
(15)TGFBI遺伝子の変異の修正が、Cas9により切断された配列の相同性配向型修復(HDR)を介して行われる、(1)~(14)のいずれかに記載の方法。
(16)HDRの鋳型ドナーとして一本鎖オリゴDNA(ssODN)が使用される、(15)に記載の方法。
(17)ssODNが、点変異を野生型のアミノ酸に相当する塩基に置き換えるノックイン配列を有する、(16)に記載の方法。
(18)野生型のアミノ酸に相当する塩基がCGT又はCGCである、(17)に記載の方法。
(19)ノックイン配列が制限酵素部位を有する、(18)に記載の方法。
(20)野生型のアミノ酸に相当する塩基がCGTであり、制限酵素部位がBsiWI部位である、(19)に記載の方法。
(21)ssODNが更に、切断部位の両端に相同的な50塩基の相同性アームをノックイン配列の両端に有する、(17)~(20)のいずれかに記載の方法。
(22)細胞との接触の際にCas9とgRNAとがリボ核タンパク質(RNP)複合体を形成している、(1)~(21)のいずれかに記載の方法。
(23)顆粒状角膜変性症に関連する、TGFBI遺伝子の変異部位を含む標的配列とハイブリダイズする、ガイドRNA分子。
(24)TGFBI遺伝子の変異が点変異である、(23)に記載のガイドRNA分子。
(25)顆粒状角膜変性症がGCD2である、(23)又は(24)に記載のガイドRNA分子。
(26)点変異がTGFBIタンパク質の124位におけるアミノ酸置換である、(23)~(25)のいずれかに記載のガイドRNA分子。
(27)アミノ酸置換がR124H、R124C及びR124Lからなる群から選択される、(26)に記載のガイドRNA分子。
(28)TGFBI遺伝子のPAM配列の上流側に隣接した22塩基の配列から成る標的配列とハイブリダイズすることができるガイド配列を含む、(23)~(28)のいずれかに記載のガイドRNA分子。
(29)PAM配列がCGGから成る、(28)に記載のガイドRNA分子。
(30)ガイド配列が、標的配列と相補的な、少なくとも17~18塩基から成る領域を含む、(28)又は(29)に記載のガイドRNA分子。
(31)ガイド配列が、アデニン又はグアニンから始まる22塩基の配列から成る、(28)~(30)のいずれかに記載のガイドRNA分子。
(32)ガイド配列が配列番号1の塩基配列を有する、(28)~(31)のいずれかに記載のガイドRNA分子。
(33)ガイド配列が、tracr RNA配列と結合している、(28)~(32)のいずれかに記載のガイドRNA分子。
(34)(23)~(33)のいずれかに記載のガイドRNA分子をコードする、核酸。
(35)(23)~(33)のいずれかに記載のガイドRNA分子を発現する、ベクター。
(36)ガイドRNAの発現がU6プロモーターによって駆動される、(35)に記載のベクター。
(37)ガイドRNAとU6プロモーターが同一又は異なるベクター上に位置する、(36)に記載のベクター。
(38)ガイドRNAとU6プロモーターが同一のベクター上で作用可能に連結している、(36)に記載のベクター。
(39)(35)~(38)のいずれかに記載のベクターと、HDRの鋳型ドナーとしてのssODN分子とを含む、キット。
(40)ssODNが、点変異を野生型のアミノ酸に相当する塩基に置き換えるノックイン配列を有する、(39)に記載のキット。
(41)野生型のアミノ酸に相当する塩基がCGT又はCGCである、(40)に記載のキット。
(42)ノックイン配列が制限酵素部位を有する、(40)又は(41)に記載のキット。
(43)野生型のアミノ酸に相当する塩基がCGTであり、制限酵素部位がBsiWI部位である、(42)に記載のキット。
(44)ssODNが更に、切断部位の両端に相同的な50塩基の相同性アームをノックイン配列の両端に有する、(39)~(43)のいずれかに記載のキット。
(45)ssODN分子が配列番号2の塩基配列を有する、(39)~(44)のいずれかに記載のキット。
(46)(23)~(33)のいずれかに記載のガイドRNA分子と、Cas9タンパク質とを含む、キット。
(47)ガイドRNA分子とCas9タンパク質とがリボ核タンパク質(RNP)複合体を形成している、(46)に記載のキット。
(48)以下のいずれか1つ以上:
(23)~(33)のいずれかに記載のガイドRNA分子;
(34)に記載のガイドRNA分子をコードする、核酸;あるいは
配列番号2の塩基配列を有する、HDRの鋳型ドナーとしてのssODN分子、
を含む医薬組成物。
一態様において、本発明は、ゲノム編集の機構を通じて、TGFBI遺伝子の変異を修復することにより顆粒状角膜変性症を治療する方法を提供する。本発明においては、顆粒状角膜変性症に関与する、あらゆるTGFBI遺伝子の変異、例えば点変異を修復することが意図される。
5′- acucagcuguacacggaccaca -3′を有するように設計される。このようなgRNAは化学的に合成することができる。
一態様において、本発明は更に、TGFBI遺伝子の変異部位を含む標的配列とハイブリダイズする、ガイドRNA分子及び/又はそれをコードする核酸、あるいはそれらの用途を提供する。ガイドRNA分子が有するガイド配列等の詳細は上述したとおりである。
pX458-hTGFBI(R124H)gRNA, RNA塩基配列(全長):
5-ACUCAGCUGUACACGGACCACAguuuuagagcuaGAAAuagcaaguuaaaauaaggcuaguccguuaucaacuugaaaaaguggcaccgagucggugcuuuu-3′
フォワード: 5′-GTTGAGTTCACGTAGACAGGC-3′(配列番号9)
リバース: 5′-GACTCCCATTCATCATGCCCA-3’(配列番号10)
Off target解析に用いたPCRプライマー一式を以下に示す。
OTS1
5′-ATGTCAGAAGTCCCGCTGTG-3′(配列番号11)
5′-TGATGGGGTCAGAGGGCATA-3′(配列番号12)
5′-CTTCCTGCTCTGTGTTTAGCCA-3′(配列番号13)
5′-ACCTCCAAGTTGAGCAGTGTC-3′(配列番号14)
5′-GCAGCAAAGCACTCAAGAGG-3′(配列番号15)
5′-CAAACTTCTGCCTGGGCATC-3′(配列番号16)
Claims (8)
- 1)顆粒状角膜変性症に関連する、トランスフォーミング増殖因子β誘発(TGFBI)遺伝子のR124変異部位を含む標的配列を標的とする、ガイドRNA分子であって、当該変異部位の2~4bp下流に位置するCGGをTGFBI遺伝子のスペーサー前隣接モチーフ(PAM)配列とし、その発現がU6プロモーターによって駆動され、
標的配列と相補的な、少なくとも17~18塩基から成る領域を含む、アデニン又はグアニンから始まる22塩基の配列から成るガイド配列であって、spCas9と複合体を形成し、それにより標的配列を切断することができるガイド配列を含む、ガイドRNA分子と、
2)R124変異を野生型のアミノ酸に相当する塩基に置き換えるノックイン配列であって、
制限酵素部位BsiWIを有するノックイン配列と、ノックイン配列の両端に、各アームが40~60塩基の相同性アームとを有するドナーDNAとを含む、
キット。 - R124変異がR124H、R124C及びR124Lからなる群から選択される、請求項1に記載のキット。
- 顆粒状角膜変性症が顆粒状角膜ジストロフィー2型(GCD2)である、請求項1又は2に記載のキット。
- ガイド配列が配列番号1の塩基配列を有し、ドナーDNAが相同性配向型修復(HDR)の鋳型ドナーとしてのssODN分子であり、ssODN分子が配列番号2の塩基配列を有する、請求項1~3のいずれか1項に記載のキット。
- ガイド配列が、trans-activating crRNA(tracr RNA)配列と結合している、請求項1~4のいずれか1項に記載のキット。
- 更にベクターを含み、ガイドRNAとU6プロモーターが当該ベクター上で作用可能に連結している、請求項1~5のいずれか一項に記載のキット。
- 野生型のアミノ酸に相当する塩基がCGT又はCGCである、請求項1~6のいずれか一項にに記載のキット。
- SpCas9を更に含む、請求項1~7のいずれか一項に記載のキット。
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Citations (7)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2015133554A1 (ja) | 2014-03-05 | 2015-09-11 | 国立大学法人神戸大学 | 標的化したdna配列の核酸塩基を特異的に変換するゲノム配列の改変方法及びそれに用いる分子複合体 |
WO2015139139A1 (en) | 2014-03-20 | 2015-09-24 | UNIVERSITé LAVAL | Crispr-based methods and products for increasing frataxin levels and uses thereof |
JP2016501036A (ja) | 2012-12-17 | 2016-01-18 | プレジデント アンド フェローズ オブ ハーバード カレッジ | Rna誘導性ヒトゲノム改変 |
WO2016021973A1 (ko) | 2014-08-06 | 2016-02-11 | 주식회사 툴젠 | 캄필로박터 제주니 crispr/cas 시스템 유래 rgen을 이용한 유전체 교정 |
WO2016210271A1 (en) | 2015-06-24 | 2016-12-29 | Sigma-Aldrich Co. Llc | Cell cycle dependent genome regulation and modification |
WO2017083852A1 (en) | 2015-11-13 | 2017-05-18 | MOORE, Tara | Methods for the treatment of corneal dystrophies |
JP2019524149A (ja) | 2016-08-20 | 2019-09-05 | アベリノ ラボ ユーエスエー インコーポレイテッドAvellino Lab USA, Inc. | 一本鎖ガイドRNA、CRISPR/Cas9システム、及びそれらの使用方法 |
Family Cites Families (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
KR100733186B1 (ko) * | 2005-05-31 | 2007-06-27 | 재단법인 목암생명공학연구소 | Hcv 유전자에 특이적인 작은 간섭 rna 및 그를유효성분으로 포함하는 c형 간염 치료제 |
WO2017004616A1 (en) * | 2015-07-02 | 2017-01-05 | The Johns Hopkins University | Crispr/cas9-based treatments |
-
2018
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- 2018-06-07 CN CN201880051724.5A patent/CN111065736A/zh active Pending
- 2018-06-07 EP EP18813909.1A patent/EP3636754A4/en active Pending
-
2019
- 2019-12-05 IL IL271216A patent/IL271216A/en unknown
Patent Citations (7)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JP2016501036A (ja) | 2012-12-17 | 2016-01-18 | プレジデント アンド フェローズ オブ ハーバード カレッジ | Rna誘導性ヒトゲノム改変 |
WO2015133554A1 (ja) | 2014-03-05 | 2015-09-11 | 国立大学法人神戸大学 | 標的化したdna配列の核酸塩基を特異的に変換するゲノム配列の改変方法及びそれに用いる分子複合体 |
WO2015139139A1 (en) | 2014-03-20 | 2015-09-24 | UNIVERSITé LAVAL | Crispr-based methods and products for increasing frataxin levels and uses thereof |
WO2016021973A1 (ko) | 2014-08-06 | 2016-02-11 | 주식회사 툴젠 | 캄필로박터 제주니 crispr/cas 시스템 유래 rgen을 이용한 유전체 교정 |
WO2016210271A1 (en) | 2015-06-24 | 2016-12-29 | Sigma-Aldrich Co. Llc | Cell cycle dependent genome regulation and modification |
WO2017083852A1 (en) | 2015-11-13 | 2017-05-18 | MOORE, Tara | Methods for the treatment of corneal dystrophies |
JP2019524149A (ja) | 2016-08-20 | 2019-09-05 | アベリノ ラボ ユーエスエー インコーポレイテッドAvellino Lab USA, Inc. | 一本鎖ガイドRNA、CRISPR/Cas9システム、及びそれらの使用方法 |
Non-Patent Citations (1)
Title |
---|
日本眼科学会雑誌,2016年,Vol.120,pp.246-263,p.259, 図18 |
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