JPWO2019131829A1 - 標的遺伝子改変用組成物 - Google Patents
標的遺伝子改変用組成物 Download PDFInfo
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- JPWO2019131829A1 JPWO2019131829A1 JP2019562142A JP2019562142A JPWO2019131829A1 JP WO2019131829 A1 JPWO2019131829 A1 JP WO2019131829A1 JP 2019562142 A JP2019562142 A JP 2019562142A JP 2019562142 A JP2019562142 A JP 2019562142A JP WO2019131829 A1 JPWO2019131829 A1 JP WO2019131829A1
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Abstract
Description
近年、ゲノム編集手段、例えばCRISPR(Clustered, regularly interspaced, short palindromic repeats)システムを利用して、様々な細胞中で遺伝子改変を行うための研究開発が進められている。しかしながら、注射等による生体への投与によって、目的とする細胞中に、CRISPRシステムで必要とされるgRNA(ガイドRNA)やRNA誘導型ヌクレアーゼ(Cas9等)をコードする遺伝子などの遺伝子改変ツールを送達し、遺伝子改変を行うことについては報告が少なく、例えば筋細胞における高い遺伝子改変効率の実現が可能な遺伝子改変ツールを送達するためのデリバリー手法の開発が望まれている。CRISPRシステムとしては、クラス1とクラス2が知られており、クラス1の中ではタイプI、タイプIII、タイプIVが、クラス2の中ではタイプII、タイプV、およびタイプVI知られている。遺伝子改変を行うにあたって、DNAに結合して切断するクラス2タイプIIのCas9が広く用いられているが、同じくDNAを結合・切断するクラス2タイプVのCpf1(Cas12a)やC2c1(Cas12b)なども利用されている。また、RNAに結合して切断するクラス2タイプVIのCas13a(C2c2)やCas13bなども報告されている。
[1]
1)式(I):
nは、2〜5の整数を、
Rは、直鎖状C1−5アルキル基、直鎖状C7−11アルケニル基又は直鎖状C11アルカジエニル基を、
波線は、それぞれ独立して、シス型またはトランス型の結合を示す。]
で表される化合物又はその塩、
2)構造脂質、並びに、
3)ガイドRNAもしくはこれをコードする配列を含むDNA、及び/又はRNA誘導型ヌクレアーゼもしくはこれをコードする配列を含む核酸を含む、細胞中の標的遺伝子座における遺伝子改変を誘導するための組成物。
[2]
RNA誘導型ヌクレアーゼがCas9であり;
ガイドRNAが、
(a)キメラRNA、又は
(b)crRNA及びtracrRNAを含む2以上のRNA
である、項1記載の組成物。
[2a]
RNA誘導型ヌクレアーゼがCpf1である、項1記載の組成物。
[3]
Cas9が化膿性レンサ球菌由来Cas9である、項1または項2記載の組成物。
[4]
前記ガイドRNAが2種類以上のガイドRNAである、項1から項3のいずれかに記載の組成物。
[5]
細胞が筋細胞である、項1から項4のいずれかに記載の組成物。
[6]
標的遺伝子座が、ジストロフィン遺伝子のヌクレオチド配列を含む、項1から項5のいずれかに記載の組成物。
[7]
ガイドRNAが、
(1)配列番号1もしくは配列番号2で示される核酸配列を含むキメラRNA、又は
(2)(i) 配列番号3もしくは配列番号4で示される核酸配列を含むcrRNA、および
(ii)配列番号7もしくは配列番号8で示される核酸配列を含むtracrRNA
である、項1から項6のいずれかに記載の組成物。
[8]
項1記載の組成物と細胞とを接触させる工程を含む、細胞中の標的遺伝子座を改変する方法。
[9]
RNA誘導型ヌクレアーゼがCas9であり;
ガイドRNAが、
(a)キメラRNA、又は
(b)crRNA及びtracrRNAを含む2以上のRNA
である、項8記載の方法。
[9a]
RNA誘導型ヌクレアーゼがCpf1である、項8記載の方法。
[10]
Cas9が化膿性レンサ球菌由来Cas9である、項8または項9記載の方法。
[11]
細胞が筋細胞である、項8から項10のいずれかに記載の方法。
[12]
標的遺伝子座が、ジストロフィン遺伝子のヌクレオチド配列を含む、項8から項11のいずれかに記載の方法。
[13]
ガイドRNAが、
(1)配列番号1もしくは配列番号2で示される核酸配列を含むキメラRNA、又は
(2)(i) 配列番号3もしくは配列番号4で示される核酸配列を含むcrRNA、および
(ii)配列番号7もしくは配列番号8で示される核酸配列を含むtracrRNA
である、項8から項12のいずれかに記載の方法。
[14]
項8から項13のいずれかに記載の方法によって得られる標的遺伝子座が改変された細胞。
[15]
項6記載の組成物を含む、医薬。
[16]
RNA誘導型ヌクレアーゼがCas9であり;
ガイドRNAが、
(a)キメラRNA、又は
(b)crRNA及びtracrRNAを含む2以上のRNA
である、項15記載の医薬。
[16a]
RNA誘導型ヌクレアーゼがCpf1である、項15記載の医薬。
[17]
Cas9が化膿性レンサ球菌由来Cas9である、項15または項16記載の医薬。
[18]
ガイドRNAが、
(1)配列番号1もしくは配列番号2で示される核酸配列を含むキメラRNA、又は
(2)(i) 配列番号3もしくは配列番号4で示される核酸配列を含むcrRNA、および
(ii)配列番号7もしくは配列番号8で示される核酸配列を含むtracrRNA
である、項15から項17のいずれかに記載の医薬。
[19]
筋ジストロフィーの予防・治療薬である、項15から項18のいずれかに記載の医薬。
[20]
修復型ジストロフィンタンパク産生薬である、項15から項19のいずれかに記載の医薬。
[21]
哺乳動物に対して項6または項7記載の組成物の有効量を投与することを特徴とする、該哺乳動物における筋ジストロフィーの予防・治療方法。
[21a]
前記投与が静脈内投与である、項21記載の予防・治療方法。
[21b]
前記投与が筋肉内投与である、項21記載の予防・治療方法。
[22]
哺乳動物に対して項6または項7記載の組成物の有効量を投与することを特徴とする、該哺乳動物における修復型ジストロフィンタンパク産生方法。
[23]
筋ジストロフィーの予防・治療剤を製造するための、項6または項7記載の組成物の使用。
[24]
筋ジストロフィーの予防・治療に使用するための、項6または項7記載の組成物。
[25]
項1から項7のいずれかに記載の組成物と細胞とを接触させる工程を含む、標的遺伝子座が改変された細胞の製造方法。
[26]
(1)項1から項7のいずれかに記載の組成物と非ヒト哺乳動物の受精卵、胚性幹細胞もしくは卵母細胞とを接触させる工程、
(2)標的遺伝子座が改変された前記受精卵、胚性幹細胞もしくは卵母細胞を選択する工程、及び
(3)前記選択された受精卵、胚性幹細胞もしくは卵母細胞を非ヒト哺乳動物の雌性動物に移植する工程
を含む、標的遺伝子座が改変された非ヒト哺乳動物の作製方法。
[27]
1)式(I):
nは、2〜5の整数を、
Rは、直鎖状C1−5アルキル基、直鎖状C7−11アルケニル基又は直鎖状C11アルカジエニル基を、
波線は、それぞれ独立して、シス型またはトランス型の結合を示す。]
で表される化合物又はその塩、および
2)構造脂質
を含む脂質粒子分散液と、
3)ガイドRNAもしくはこれをコードする配列を含むDNA、及び/又はRNA誘導型ヌクレアーゼもしくはこれをコードする配列を含む核酸
を含む水溶液とを混合する工程を含む、
細胞中の標的遺伝子座における遺伝子改変を誘導するための組成物の製造方法。
[28]
前記ガイドRNAが2種類以上のガイドRNAである、項27記載の製造方法。
nは、好ましくは3〜5の整数であり、より好ましくは3である。
波線は、好ましくは両方ともシス型の結合である。
化合物(A):nが3〜5の整数であり、Rがシス型の直鎖状C7−11アルケニル基であり、波線が両方ともシス型となる結合である化合物。
化合物(B):nが4であり、Rが、2つの炭素−炭素二重結合の両方においてシス型の、直鎖状C11アルカジエニル基であり、波線が両方ともシス型の結合である化合物。
化合物(C):nが2または3であり、Rが直鎖状C1−5アルキル基であり、波線が両方ともシス型の結合である化合物。
化合物(A1):nが3〜5の整数であり、Rがシス型の5−ヘプテニル、7−ノネニルまたは9−ウンデセニルであり、波線が両方ともシス型となる結合である化合物。
化合物(B1):nが4であり、Rが、2つの炭素−炭素二重結合の両方においてシス型の、2,5−ウンデカジエニルであり、波線が両方ともシス型の結合である化合物。
化合物(C1):nが2または3であり、Rがメチル、プロピル、またはペンチルであり、波線が両方ともシス型の結合である化合物。
ステロール類(例えば、コレステロール、コレステロールエステル、コレステロールヘミコハク酸など);
リン脂質(例えば、ホスファチジルコリン(例えば、ジパルミトイルホスファチジルコリン、ジステアロイルホスファチジルコリン、リソホスファチジルコリン、ジオレオイルホスファチジルコリン、パルミトイルオレオイルホスファチジルコリン、ジリノレノイルホスファチジルコリン、MC−1010(NOF CORPORATION)、MC−2020(NOF CORPORATION)、MC−4040(NOF CORPORATION)など)、ホスファチジルセリン(例えば、ジパルミトイルホスファチジルセリン、ジステアロイルホスファチジルセリン、ジオレオイルホスファチジルセリン、パルミトイルオレオイルホスファチジルセリンなど)、ホスファチジルエタノールアミン(例えば、ジパルミトイルホスファチジルエタノールアミン、ジステアロイルホスファチジルエタノールアミン、ジオレオイルホスファチジルエタノールアミン、パルミトイルオレオイルホスファチジルエタノールアミン、リソホスファチジルエタノールアミンなど)、ホスファチジルイノシトール、ホスファチジン酸など);および
ポリエチレングリコール脂質(PEG脂質)(例えば、PEG−DAA、PEG−DAG、PEG−phospholipid cunjugate、PEG−Cer、PEG−cholesterol、PEG−C−DOMG、2KPEG−CMG、GM−020(NOF CORPORATION)、GS−020(NOF CORPORATION)、GS−050(NOF CORPORATION)など)
からなる群より選ばれる少なくとも1種を用いることができる。本発明では、構造脂質として、ステロール類(特に、コレステロール)、リン脂質(特に、ホスファチジルコリン)およびポリエチレングリコール脂質の3種全てを用いることが好ましい。
5'- U(M)^G(M)^G(M)^UAUCUUACAGGAACUCCGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGG(M)^U(M)^G(M)^C -3'
配列番号2:実施例「HsDMDEx45#23」対応sgRNA全配列
5'- A(M)^G(M)^C(M)^UGUCAGACAGAAAAAAGGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGG(M)^U(M)^G(M)^C -3'
配列番号3:配列番号1の標的認識配列
5'- U(M)^G(M)^G(M)^UAUCUUACAGGAACUCC -3'
配列番号4:配列番号2の標的認識配列
5'- A(M)^G(M)^C(M)^UGUCAGACAGAAAAAAG -3'
配列番号5:配列番号1のcrRNA配列
5'- U(M)^G(M)^G(M)^UAUCUUACAGGAACUCCGUUUUAGAGCUA -3'
配列番号6:配列番号2のcrRNA配列
5'- A(M)^G(M)^C(M)^UGUCAGACAGAAAAAAGGUUUUAGAGCUA -3'
配列番号7:配列番号1のtracrRNA配列
5'- UAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGG(M)^U(M)^G(M)^C -3'
配列番号8:配列番号2のtracrRNA配列
5'- UAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGG(M)^U(M)^G(M)^C -3'
配列番号1〜8において、「(M)」が右隣に示されているリボースは、天然の(修飾されていない)リボースであってもよいし、2’−O−メチルリボースまたはその他の修飾リボースであってもよいが、好ましくは2’−O−メチルリボースである。
また、配列番号1〜8において「^」で表されている、2’−O−メチルリボース同士の結合または2’−O−メチルリボースとリボースとの結合は、リン酸ジエステル結合であってもよいし、ホスホロチオエート結合であってもよいが、好ましくはホスホロチオエート結合である。
エーテル類:ジエチルエーテル、ジイソプロピルエーテル、ジフェニルエーテル、テトラヒドロフラン、1,2−ジメトキシエタンなど;
芳香族炭化水素類:クロロベンゼン、トルエン、キシレンなど;
飽和炭化水素類:シクロヘキサン、ヘキサン、ヘプタンなど;
アミド類:N,N−ジメチルホルムアミド、N−メチルピロリドンなど;
ハロゲン化炭化水素類:ジクロロメタン、四塩化炭素など;
ニトリル類:アセトニトリルなど;
スルホキシド類:ジメチルスルホキシドなど;
芳香族有機塩基類:ピリジンなど;
酸無水物類:無水酢酸など;
有機酸類:ギ酸、酢酸、トリフルオロ酢酸など;
無機酸類:塩酸、硫酸など;
エステル類:酢酸エチル、酢酸イソプロピルエステルなど;
ケトン類:アセトン、メチルエチルケトンなど;
水。
上記溶媒は、二種以上を適宜の割合で混合して用いてもよい。
塩基性塩類:炭酸ナトリウム、炭酸カルシウム、炭酸水素ナトリウムなど;
有機塩基類:トリエチルアミン、ジエチルアミン、ピリジン、4−ジメチルアミノピリジン、N,N−ジメチルアニリン、1,4−ジアザビシクロ[2.2.2]オクタン、1,8−ジアザビシクロ[5.4.0]−7−ウンデセン、イミダゾール、ピペリジンなど;
金属アルコキシド類:ナトリウムエトキシド、カリウムtert−ブトキシド、ナトリウムtert−ブトキシドなど;
アルカリ金属水素化物類:水素化ナトリウムなど;
金属アミド類:ナトリウムアミド、リチウムジイソプロピルアミド、リチウムヘキサメチルジシラジドなど;
有機リチウム類:n−ブチルリチウム、sec−ブチルリチウムなど。
有機酸類:酢酸、トリフルオロ酢酸、クエン酸、p−トルエンスルホン酸、10−カンファースルホン酸など;
ルイス酸:三フッ化ホウ素ジエチルエーテル錯体、ヨウ化亜鉛、無水塩化アルミニウム、無水塩化亜鉛、無水塩化鉄など。
MS:マススペクトル
M:モル濃度
N:規定度
CDCl3:重クロロホルム
DMSO−d6:重ジメチルスルホキシド
1H NMR:プロトン核磁気共鳴
LC/MS:液体クロマトグラフ質量分析計
ESI:electrospray ionization、エレクトロスプレーイオン化
APCI:atmospheric pressure chemical ionization、大気圧化学イオン化
MALDI:Matrix-assisted laser desorption/ionization、マ卜リックス支援レーザー脱離イオン化
TOFMS:Time-of-flight mass spectrometry、飛行時間型質量分析
CHCA:α-シアノ-4-ヒドロキシケイ皮酸
DMF:N,N-ジメチルホルムアミド
THF:テトラヒドロフラン
DMAP:4−ジメチルアミノビリジン
TBAF:テトラブチルアンモニウムフルオリド
A) 2-(((tert-ブチルジメチルシリル)オキシ)メチル)-2-(ヒドロキシメチル)プロパン-1,3-ジオール
2, 2-ビス(ヒドロキシメチル)プロパン-1,3-ジオール(5.45 g)、1H-イミダゾール(2.72 g)およびDMF(190 mL)の混合物に、tert-ブチルクロロジメチルシラン(3.01 g)のDMF(10 mL)溶液を室温で加えた。24時間撹拌後、反応混合物を減圧下濃縮した。残渣を酢酸エチルで希釈し、水で3回、飽和食塩水で1回洗浄後、無水硫酸ナトリウムで乾燥し、溶媒を減圧下留去した。残渣をシリカゲルカラムクロマトグラフィー(酢酸エチル/ヘキサン)で精製して標題化合物(2.25 g)を得た。
1H NMR (300 MHz, CDCl3) δppm 0.08 (6H, s), 0.90 (9H, s), 2.53 (3H, t, J = 5.5 Hz), 3.66 (2H, s), 3.73 (6H, d, J = 5.5 Hz)
2-(((tert-ブチルジメチルシリル)オキシ)メチル)-2-(ヒドロキシメチル)プロパン-1,3-ジオール(258 mg)、(9Z)-テトラデカ-9-エン酸(769 mg) および DMAP (126 mg)のDMF(3 mL)溶液に1-エチル-3-(3-ジメチルアミノプロピル)カルボジイミド塩酸塩(790 mg)を室温で加えた。18時間撹拌後、反応混合物を酢酸エチルで希釈し、水で2回、飽和食塩水で1回洗浄後、無水硫酸ナトリウムで乾燥し、溶媒を減圧下留去した。残渣をシリカゲルカラムクロマトグラフィー(NH、酢酸エチル/ヘキサン)で精製して標題化合物(860 mg)を得た。
1H NMR (300 MHz, CDCl3) δppm 0.03 (6H, s), 0.81-0.96 (18H, m), 1.18-1.41 (36H, m), 1.53-1.67 (6H,m), 1.91-2.10 (12H, m), 2.29 (6H, t, J = 7.6 Hz), 3.58 (2H, s), 4.08 (6H, s), 5.27-5.43 (6H, m)
3-((tert-ブチル(ジメチル)シリル)オキシ)-2,2-ビス(((9Z)-テトラデカ-9-エノイルオキシ)メチル)プロピル(9Z)-テトラデカ-9-エノアート(5.91 g)のTHF(120 mL)溶液に、TBAFのTHF溶液(1 M, 14.85 mL)と酢酸(4.91 mL)の混合物を室温で加えた。3日間撹拌後、反応混合物を減圧下濃縮した。残渣を酢酸エチルで希釈し、飽和炭酸水素ナトリウム水溶液で1回、飽和食塩水で1回洗浄後、無水硫酸ナトリウムで乾燥し、溶媒を減圧下留去した。残渣をシリカゲルカラムクロマトグラフィー(酢酸エチル/ヘキサン)で精製して標題化合物(4.96 g)を得た。
1H NMR (300 MHz, CDCl3) δppm 0.82-0.97 (9H, m), 1.16-1.42 (36H, m), 1.52-1.68 (6H, m), 1.90-2.12 (12H, m), 2.32 (6H, t, J = 7.6 Hz), 2.52 (1H, t, J = 7.0 Hz), 3.49 (2H, d, J = 7.0 Hz), 4.11 (6H, s), 5.26-5.42 (6H, m)
3-ヒドロキシ-2,2-ビス(((9Z)-テトラデカ-9-エノイルオキシ)メチル)プロピル(9Z)-テトラデカ-9-エノアート(4.96 g)、DMAP (796 mg)および4-(ジメチルアミノ)ブタン酸塩酸塩(2.19 g)のDMF(20 mL)溶液に1-エチル-3-(3-ジメチルアミノプロピル)カルボジイミド塩酸塩(2.50 g)を室温で加えた。18時間撹拌後、反応混合物を酢酸エチルで希釈し、飽和炭酸水素ナトリウム水溶液で1回、飽和食塩水で1回洗浄後、無水硫酸ナトリウムで乾燥し、溶媒を減圧下留去した。残渣をシリカゲルカラムクロマトグラフィー(NH、酢酸エチル/ヘキサン)で精製して標題化合物(5.31 g)を得た。
1H NMR (300 MHz, CDCl3) δppm 0.82-0.94 (9H, m), 1.20-1.42 (36H, m), 1.50-1.66 (6H, m), 1.69-1.83 (2H, m), 1.90-2.10 (12H, m), 2.20 (6H, s), 2.23-2.41 (10H, m), 4.11 (8H, s), 5.23-5.44 (6H, m)
脂質混合物(合成例1で得られたカチオン性脂質:DPPC:コレステロール:GM−020=60:10.6:28.7:0.7,モル比)を、90% EtOH,10% RNaseフリー水に溶解して6.9mg/mLの脂質溶液を得た。Cas9 mRNA(TriLink BioTechnologies)を、10 mM 2−MES溶液pH5.5に溶解して0.15 mg/mLの核酸溶液を得た。得られた脂質溶液および核酸溶液を、室温で、NanoAssemblr(Precision NanoSystems)によって流速比2.7 mL/min:5.3 mL/minで混合し、核酸を内封する脂質粒子を含む分散液を得た。得られた分散液は、Slyde−A−Lyzer(20kの分画分子量、Thermo scientific)を用いて、水に対して4℃で1時間、PBSに対して4℃で18時間透析を行った。さらにAmicon(30kの分画分子量)を用いて遠心(3,000×g,4℃,一定体積に落ちるまで20分を数回)し、限外ろ過による濃縮を行った。続いて、0.2 μmのsyringe filter(Iwaki)を用いてフィルターろ過を行い、4℃に保存した。このようにして調製した分散液を「Cas9 mRNA−LNP」として後記試験で用いた。脂質粒子の粒子径および多分散指数(PDI)はZetasizer Nano ZS(Malvern Instruments)によって測定した。また、脂質粒子における核酸の濃度および内封率はQuant−iTTM RiboGreen(登録商標)(Thermo scientific)を用いて測定した。測定結果を表2に示す。
sgRNA−LNPの調製
脂質混合物(合成例1で得られたたカチオン性脂質:DPPC:コレステロール:GM−020=60:10.6:28.7:0.7,モル比)を、90% EtOH,10%RNaseフリー水に溶解して6.9 mg/mLの脂質溶液を得た。MmRosa26 sgRNA(GeneDesign、前記表1参照)を、10 mM 2−MES溶液pH5.5に溶解して0.15 mg/mLの核酸溶液を得た。得られた脂質溶液および核酸溶液を、室温で、NanoAssemblr(Precision NanoSystems)によって流速比2.7 mL/min:5.3 mL/minで混合し、核酸を内封する脂質粒子を含む分散液を得た。得られた分散液は、Slyde−A−Lyzer(20kの分画分子量、Thermo scientific)を用いて、水に対して4℃で1時間、PBSに対して4℃で18時間透析を行った。さらにAmicon(30kの分画分子量)を用いて遠心(3,000×g,4℃,一定体積に落ちるまで20分を数回)し、限外ろ過による濃縮を行った。続いて、0.2 μmのsyringe filter(Iwaki)を用いてフィルターろ過を行い、4℃に保存した。このようにして調製した分散液を「MmRosa26 sgRNA−LNP」として後記試験で用いた。脂質粒子の粒子径および多分散指数(PDI)はZetasizer Nano ZS(Malvern Instruments)によって測定した。また、脂質粒子における核酸の濃度および内封率はQuant−iTTM RiboGreen(登録商標)(Thermo scientific)を用いて測定した。測定結果を表2に示す。
9週齡の雄性C57BL/6Jマウス(日本クレア)の右下肢腓腹筋に、MmRosa26 sgRNA−LNPおよびCas9 mRNA−LNPの混合溶液(sgRNAおよびmRNAとしての投与量が共に0.01、0.03、0.1、0.3又は1 μg/マウスとなるよう、それぞれのLNP分散液を混合して調製したもの)、またはPBSを投与した。投与4日後、3.5%イソフルラン麻酔下で断頭放血にて安楽死させた後、骨格筋を摘出し液体窒素で速やかに凍結させた。凍結筋組織からQIAamp Fast DNA Tissue Kit(Qiagen)を用いてゲノムDNAを抽出精製し、PrimeSTAR GXL DNA polymerase(TAKARA)を用いてPCR(Forward primer;配列番号10、Reverse primer;配列番号11)を行った。PCR産物をQIAquick PCR purification kit(QIAGEN)で精製し、T7 Endonuclease I(NEB)で処理後、Agilent 4200 TapeStation(Agilent)を用いて解析した。結果を図1に示す。
配列番号10
5'- CTCCGAGGCGGATCACAAGCAATAATAACCTGTAG -3'
配列番号11
5'- TGCAAGCACGTTTCCGACTTGAGTTGCCTCAAGAG -3'
実施例1の[1−1]に記載した手順で再度Cas9 mRNA−LNPを調製し、核酸濃度、内封率、粒子径およびPDIを測定した。測定結果を表3に示す。
脂質混合物(合成例1で製造したカチオン性脂質:DPPC:コレステロール:GM−020=60:10.6:28.7:0.7,モル比)を、90% EtOH,10% RNaseフリー水に溶解して6.9 mg/mLの脂質溶液を得た。HsDMDEx45#1 sgRNA(GeneDesign、前記表1参照)を、10 mM 2−モルホリノエタンスルホン酸(MES)溶液pH5.5に溶解して0.15 mg/mLの核酸溶液を得た。得られた脂質溶液および核酸溶液を、室温で、NanoAssemblr(Precision NanoSystems)によって流速比2.7 mL/min:5.3 mL/minで混合し、核酸を内封する脂質粒子を含む分散液を得た。得られた分散液は、Slyde−A−Lyzer(20kの分画分子量、Thermo scientific)を用いて、水に対して4℃で1時間、PBSに対して4℃で18時間透析を行った。さらにAmicon(30kの分画分子量)を用いて遠心(3,000×g,4℃,一定体積に落ちるまで20分を数回)し、限外ろ過による濃縮を行った。続いて、0.2 μmのsyringe filter(Iwaki)を用いてフィルターろ過を行い、4℃に保存した。このようにして調製した分散液を「HsDMDEx45#1 sgRNA−LNP」として後記試験で用いた。脂質粒子の粒子径および多分散指数(PDI)はZetasizer Nano ZS(Malvern Instruments)によって測定した。また、脂質粒子における核酸の濃度および内封率はQuant−iTTM RiboGreen(登録商標)(Thermo scientific)を用いて測定した。測定結果を表3に示す。
44ノックアウトマウスの作製方法
10 μgのヒトDMD エクソン45およびその5’側0.7 kb、3’側0.6 kbを含む配列1.5kb、FRT配列で挟まれたネオマイシン耐性遺伝子発現ユニットおよびマウスDmd イントロン 44とイントロン 45由来配列各1.5 kbからなるノックインベクターを、2.5 μgのpCAG−Cas9発現ベクターおよび2種類の2.5 μgのpU6−sgRNA発現ベクター(ターゲット配列;配列番号12および配列番号13)とともに、5×105個のC57BL/6Jマウス由来のES細胞にエレクトロポレーションし、PCRおよびシーケンス確認により相同組み換え細胞株を選抜した。Flpe(フリッパーゼ)処理によりネオマイシン耐性ユニットを除去した後、当該ES細胞株をICRマウスの4倍体胚盤胞に顕微注入し、キメラマウスを取得した。キメラマウスと雌性C57BL/6Jマウスとの体外受精により雌性ヒトDMD エクソン45ヘテロノックインマウスを取得した。続いて、雄性C57BL/6Jマウスと雌性ヒトDMD エクソン45ヘテロノックインマウスの受精卵に100 ng/μLのCas9 mRNA(TriLink BioTechnologies)、2種類のマウスDmd エクソン 44ノックアウト用sgRNA(ターゲット配列;配列番号14および配列番号15、株式会社FASMAC)およびssODN 50 ng/μL(配列番号16、ユーロフィンジェノミクス株式会社)を顕微注入し、得られた雄性産仔について、PCRおよびシーケンス確認による遺伝子判定を行い、ヒトDMD エクソン45ノックイン−マウスDmd エクソン44ノックアウトマウスを選抜した。
配列番号12
5’- atgaatgtgcctacatatgg -3’
配列番号13
5’- catagcatgcatttggcttc -3’
配列番号14
5’- gaatgaggtagtgttgtagg -3’
配列番号15
5’- gcaggaaatcatcttatagc -3’
配列番号16
5’- gagcaagctgggttagaacaaaggtctgtcagagtcagcatgggaatgaggtagtgttgtagcaggaaatagtgtggtttaggtctctccccgccctctgtgtatgtgtgtgtgtgtgtt -3’
12週齡の雄性ヒトDMD エクソン45ノックイン−マウスDmd エクソン44ノックアウトマウスの右下肢腓腹筋に、3 μgのHsDMD Ex45#1 sgRNAを内封したLNPと3 μgのSpCas9 mRNAを内封したLNP([2−1]のCas9 mRNA−LNPと[2−2]のHsDMDEx45#1 sgRNA−LNPの混合溶液)を、左下肢腓腹筋にはPBSを、それぞれ6回/2週間投与した。初回投与から56日後に、3.5%イソフルラン麻酔下で安楽死させた後、骨格筋を摘出し液体窒素で速やかに凍結させた。凍結筋組織を3% protenase inhibitor cocktail(Sgima)と5 mM EDTA(WAKO)を含有したRIPA buffer(WAKO)でホモジナイズ後、QIAamp Fast DNA Tissue Kit(Qiagen)を用いてゲノムDNAを抽出精製し、PrimeSTAR GXL DNA polymerase(TAKARA)と配列番号17(Forward primer)および配列番号18(Reverse primer)のprimer setを用いて増幅した。PCR産物をQIAquick PCR purification kit(QIAGEN)で精製し、再アニーリングした後にT7 Endonuclease I(NEB)で処理し、Agilent 4200 TapeStation(Agilent)を用いて電気泳動し、付属のソフトウェアーで解析した。得られた数値を用いて、変異導入効率を下記の計算式(数1)で求めた。
配列番号17
5’- CAAGTTTAAAATAGCAGAAAACCACTAACTAGCCA -3’
配列番号18
5’- CTGACACATAAAAGGTGTCTTTCTGTCTTGTATCC -3’
ホモジネートの一部にQIAzol Lysis Reagent(QIAGEN)とクロロフォルム(WAKO)を添加し、混合・遠心後にRNAを含む水槽を分離回収し、RNeasy Mini Kit(QIAGEN)を用いて、total RNAを抽出精製した。Total RNAをHigh Capacity RNA−to−cDNA kit(Thermo)を用いて逆転写し、続けてPrimeSTAR GXL DNA polymerase(TAKARA)と配列番号19(Forward primer)および配列番号20(Reverse primer)のprimer setを用いて増幅させた。PCR産物をQIAquick PCR purification kit(QIAGEN)で精製後、Agilent 4200 TapeStation(Agilent)を用いて電気泳動し、付属のソフトウェアーで解析した。得られた数値を用いてエクソンスキッピング効率(exon skipping efficiency)を下記の計算式(数2)で求めた。
配列番号19
5’- GGTGAAAGTACAGGAAGCCGT -3’
配列番号20
5’- TTAGCTGCTGCTCATCTCCAA -3’
ホモジネートの一部を遠心し、上清を回収した。上清中の総タンパク質量をProtein assay kit(Thermo)で測定し、0.02 μg/uL及び3 μg/uLに調整した。
実施例1の[1−1]に記載した手順で再度Cas9 mRNA−LNPを調製し、核酸濃度、内封率、粒子径およびPDIを測定した。測定結果を表4に示す。
実施例2の[2−2]で作製したHsDMDEx45#1 sgRNA−LNPを再び使用した。実施例2のときの測定結果を表4に再掲する。
Doxycycline誘導型MyoD発現カセットを含むジストロフィンEx 45欠損DMD患者由来のヒトiPS細胞を、10 μM Y−27632を含むAK02N培地(Ajinomoto)に懸濁し、マトリゲルがコートされた6 well plateに3×105 cells/wellの密度で播種した。翌日、培地をPrimate ES Cell Medium(Reprocell)へと交換し、さらにその翌日に1 μg/mL doxycyclineを含むPrimate ES Cell培地に交換することによりMyoD遺伝子発現誘導を開始した。doxycycline添加から24時間後、5% KSRと1 μg/mL doxycyclineを含むalpha Minimal Essential Medium(SIGMA)培地に交換し、3日間培養した。培地を700 μLに減らし、Cas9 mRNA−LNP(mRNAとして1,3又は10μg/well)およびHsDMDEx45#1 sgRNA−LNP(sgRNAとして1,3,10 μg/well)の混合物を添加した。添加6時間後に、1.3 mLの培地(alpha Minimal Essential Medium,5% KSR,1μg/mL doxycycline)を加えた。
LNPの添加から72時間後、細胞を回収し、QIAamp DNA mini kit(Qiagen)を用いてDNAを抽出精製した。PrimeSTAR GXL DNA polymerase(TAKARA)を用いたPCR(Forward primer:前記配列番号17, Reverse primer:前記配列番号18)によって標的配列を含むゲノム領域を増幅し、得られたPCR産物をQIAquick PCR purification kit(Qiagen)によって精製した。精製したDNA断片を再アニーリングした後にT7 Endonuclease I(NEB)で処理し、Agilent 4200 TapeStation(Agilent)を用いて電気泳動し、付属のソフトウェアーで解析した。変異導入効率の計算式は[2−4]と同様である(前記数1参照)。結果を図5に示す。
回収した細胞から、RNA easy mini kit(Qiagen)を用いてtotal RNAを抽出精製した。Total RNAをHigh Capacity RNA−to−cDNA kit(Thermo)を用いて逆転写し、PrimeSTAR GXL DNA polymerase(TAKARA)を用いてPCR(Forward primer:配列番号21, Reverse primer:配列番号22)を行った。得られたPCR産物をQIAquick PCR purification kit(Qiagen)によって精製し、Agilent 4200 TapeStationを用いて電気泳動し、付属のソフトウェアーで解析した。エクソンスキッピング効率(exon skipping efficiency)の計算式は[2−5]と同様である(前記数2参照)。結果を図6に示す。
配列番号21
5’- CTACAGGAAGCTCTCTCCCA -3’
配列番号22
5’- TGCTTCCTCCAACCATAAAACA -3’
実施例1の[1−1]に記載した手順で再度Cas9 mRNA−LNPを調製し、核酸濃度、内封率、粒子径およびPDIを測定した。測定結果を表5に示す。
実施例2の[2−2]で作製したHsDMDEx45#1 sgRNA−LNPを再び使用した。実施例2のときの測定結果を表5に再掲する。
実施例2の[2−2]に記載した手順において、「HsDMDEx45#1 sgRNA」(実施例4におけるHsDMDEx45#1 sgRNA、前記表1参照)の代わりに「HsDmdEx45#23 sgRNA」(前記表1の「HsDMDEx45#23 sgRNA」参照)を用いたこと以外は同様にして、HsDMDEx45#23 sgRNA−LNPを調製し、核酸濃度、内封率、粒子径およびPDIを測定した。測定結果を表5に示す。
Doxycycline誘導型MyoD発現カセットを含むジストロフィンEx 45欠損DMD患者由来のヒトiPS細胞および健常人由来ヒトiPS細胞を、10 μM Y−27632を含むAK02N培地(Ajinomoto)に懸濁し、マトリゲルがコートされた6 well plateに1×105 cells/wellの密度で播種した。翌日、培地をPrimate ES Cell Medium(Reprocell)へと交換し、さらにその翌日に1 μg/mL doxycyclineを含む培地(Primate ES Cell Mediumに交換することによりMyoD遺伝子発現誘導を開始した。doxycycline添加から24時間後、5% KSRと1 μg/mL doxycyclineを含むalpha Minimal Essential Medium(SIGMA)培地に交換、3日間培養した。患者由来ヒトiPS細胞の培地を700 μLに減らし、LNPの混合物を添加した(下記表6参照)。添加6時間後に、1.3 mLの培地(alpha Minimal Essential Medium,5% KSR,1μg /mL doxycycline)を加えた。
LNP添加から72時間後に、冷却されたPBSで各wellを2回洗浄後、セルスクレーパーで細胞を回収し、4 ℃ 15,000 rpmで5分間遠心分離した。その後、上清を取り除きRIPA bufferで細胞を溶解した。細胞溶解液の一部から、RNA easy mini kit(Qiagen)を用いてtotal RNAを抽出精製した。Total RNAをHigh Capacity RNA−to−cDNA kit(Thermo)を用いて逆転写し、PrimeSTAR GXL DNA polymerase(TAKARA)を用いてPCR (Forward primer:前記配列番号21, Reverse primer:前記配列番号22)を行った。得られたPCR産物をQIAquick PCR purification kit(Qiagen)によって精製し、Agilent 4200 TapeStationを用いて電気泳動し、付属のソフトウェアーで解析した。エクソンスキッピング効率(exon skipping efficiency)の計算式は[2−5]と同様である(前記数2参照)。結果を図7に示す。
細胞溶解液の一部の総タンパク質量をProtein assay kit(Thermo)で測定し、0.083 μg/μL及び0.58 μg/μLに調製した。
脂質混合物(合成例1で得られたカチオン性脂質:DPPC:コレステロール:GM−020=60:10.6:28.7:0.7,モル比)を、90% EtOH,10% RNaseフリー水に溶解して6.9 mg/mLの脂質溶液を得た。Cas9 mRNA(TriLink BioTechnologies)を、10 mM 2−MES溶液pH5.5に溶解して0.15 mg/mLの核酸溶液を得た。得られた脂質溶液および核酸溶液を、室温で、NanoAssemblr(Precision NanoSystems)によって流速比2.7 mL/min:5.3 mL/minで混合し、核酸を内封する脂質粒子を含む分散液を得た。得られた分散液は、Slyde−A−Lyzer(20kの分画分子量、Thermo scientific)を用いて、水に対して4℃で1時間、PBSに対して4℃で18時間透析を行った。さらにAmicon(30kの分画分子量)を用いて遠心(3,000×g,4℃,一定体積に落ちるまで20分を数回)し、限外ろ過による濃縮を行った。続いて、0.2 μmのsyringe filter(Iwaki)を用いてフィルターろ過を行い、4℃に保存した。このようにして調製した分散液を「Cas9 mRNA−LNP」として後記試験で用いた。脂質粒子の粒子径および多分散指数(PDI)はZetasizer Nano ZS(Malvern Instruments)によって測定した。また、脂質粒子における核酸の濃度および内封率はQuant−iTTM RiboGreen(登録商標)(Thermo scientific)を用いて測定した。結果を表7に示す。
脂質混合物(合成例1で得られたカチオン性脂質:DPPC:コレステロール:GM−020=60:10.6:28.7:0.7,モル比)を、90% EtOH,10% RNaseフリー水に溶解して6.9 mg/mLの脂質溶液を得た。MmRosa26 sgRNA(GeneDesign、前記表1参照)を、10 mM 2−MES溶液pH5.5に溶解して0.15 mg/mLの核酸溶液を得た。得られた脂質溶液および核酸溶液を、室温で、NanoAssemblr(Precision NanoSystems)によって流速比2.7 mL/min:5.3 mL/minで混合し、核酸を内封する脂質粒子を含む分散液を得た。得られた分散液は、Slyde−A−Lyzer(20kの分画分子量、Thermo scientific)を用いて、水に対して4℃で1時間、PBSに対して4℃で18時間透析を行った。さらにAmicon(30kの分画分子量)を用いて遠心(3,000×g,4℃,一定体積に落ちるまで20分を数回)し、限外ろ過による濃縮を行った。続いて、0.2 μmのsyringe filter(Iwaki)を用いてフィルターろ過を行い、4℃に保存した。このようにして調製した分散液を「MmRosa26 sgRNA−LNP」として後記試験で用いた。脂質粒子の粒子径および多分散指数(PDI)はZetasizer Nano ZS(Malvern Instruments)によって測定した。また、脂質粒子における核酸の濃度および内封率はQuant−iTTM RiboGreen(登録商標)(Thermo scientific)を用いて測定した。結果を表7に示す。
[6−1]Cas9 mRNA−LNPの調製
脂質混合物(合成例1で得られたカチオン性脂質:DPPC:コレステロール:GM−020=60:10.6:28.7:0.7,モル比)を、90% EtOH,10% RNaseフリー水に溶解して6.9mg/mLの脂質溶液を得た。Cas9 mRNA(TriLink BioTechnologies)を、10 mM 2−MES溶液pH5.5に溶解して0.15 mg/mLの核酸溶液を得た。得られた脂質溶液および核酸溶液を、室温で、NanoAssemblr(Precision NanoSystems)によって流速比2.7 mL/min:5.3 mL/minで混合し、核酸を内封する脂質粒子を含む分散液を得た。得られた分散液は、Slyde−A−Lyzer(20kの分画分子量、Thermo scientific)を用いて、水に対して4℃で1時間、PBSに対して4℃で18時間透析を行った。さらにAmicon(30kの分画分子量)を用いて遠心(3,000×g,4℃,一定体積に落ちるまで20分を数回)し、限外ろ過による濃縮を行った。続いて、0.2 μmのsyringe filter(Iwaki)を用いてフィルターろ過を行い、4℃に保存した。このようにして調製した分散液を「Cas9 mRNA−LNP」として後記試験で用いた。脂質粒子の粒子径および多分散指数(PDI)はZetasizer Nano ZS(Malvern Instruments)によって測定した。また、脂質粒子における核酸の濃度および内封率はQuant−iTTM RiboGreen(登録商標)(Thermo scientific)を用いて測定した。結果を表8に示す。
脂質混合物(合成例1で製造したカチオン性脂質:DPPC:コレステロール:GM−020=60:10.6:28.7:0.7,モル比)を、90% EtOH,10% RNaseフリー水に溶解して6.9 mg/mLの脂質溶液を得た。HsDMDEx45#1 sgRNAおよびHsDMDEx45#23 sgRNAを、10 mM 2−モルホリノエタンスルホン酸(MES)溶液pH5.5に等量ずつ溶解して0.15 mg/mLの核酸溶液を得た。得られた脂質溶液および核酸溶液を、室温で、NanoAssemblr(Precision NanoSystems)によって流速比2.7 mL/min:5.3 mL/minで混合し、核酸を内封する脂質粒子を含む分散液を得た。得られた分散液は、Slyde−A−Lyzer(20kの分画分子量、Thermo scientific)を用いて、水に対して4℃で1時間、PBSに対して4℃で18時間透析を行った。さらにAmicon(30kの分画分子量)を用いて遠心(3,000×g,4℃,一定体積に落ちるまで20分を数回)し、限外ろ過による濃縮を行った。続いて、0.2 μmのsyringe filter(Iwaki)を用いてフィルターろ過を行い、4℃に保存した。このようにして調製した分散液を「HsDMDEx45#1+#23 sgRNA−LNP」として後記試験で用いた。脂質粒子の粒子径および多分散指数(PDI)はZetasizer Nano ZS(Malvern Instruments)によって測定した。また、脂質粒子における核酸の濃度および内封率はQuant−iTTM RiboGreen(登録商標)(Thermo scientific)を用いて測定した。結果を表8に示す。
Claims (28)
- RNA誘導型ヌクレアーゼがCas9であり;
ガイドRNAが、
(a)キメラRNA、又は
(b)crRNA及びtracrRNAを含む2以上のRNA
である、請求項1記載の組成物。 - Cas9が化膿性レンサ球菌由来Cas9である、請求項2記載の組成物。
- 前記ガイドRNAが2種類以上のガイドRNAである、請求項1記載の組成物。
- 細胞が筋細胞である、請求項1記載の組成物。
- 標的遺伝子座が、ジストロフィン遺伝子のヌクレオチド配列を含む、請求項5記載の組成物。
- ガイドRNAが、
(1)配列番号1もしくは配列番号2で示される核酸配列を含むキメラRNA、又は
(2)(i) 配列番号3もしくは配列番号4で示される核酸配列を含むcrRNA、および
(ii)配列番号7もしくは配列番号8で示される核酸配列を含むtracrRNA
である、請求項5記載の組成物。 - 請求項1記載の組成物と細胞とを接触させる工程を含む、細胞中の標的遺伝子座を改変する方法。
- RNA誘導型ヌクレアーゼがCas9であり;
ガイドRNAが、
(a)キメラRNA、又は
(b)crRNA及びtracrRNAを含む2以上のRNA
である、請求項8記載の方法。 - Cas9が化膿性レンサ球菌由来Cas9である、請求項9記載の方法。
- 細胞が筋細胞である、請求項8記載の方法。
- 標的遺伝子座が、ジストロフィン遺伝子のヌクレオチド配列を含む、請求項11記載の方法。
- ガイドRNAが、
(1)配列番号1もしくは配列番号2で示される核酸配列を含むキメラRNA、又は
(2)(i) 配列番号3もしくは配列番号4で示される核酸配列を含むcrRNA、および
(ii)配列番号7もしくは配列番号8で示される核酸配列を含むtracrRNA
である、請求項11記載の方法。 - 請求項8記載の方法によって得られる標的遺伝子座が改変された細胞。
- 請求項6記載の組成物を含む、医薬。
- RNA誘導型ヌクレアーゼがCas9であり;
ガイドRNAが、
(a)キメラRNA、又は
(b)crRNA及びtracrRNAを含む2以上のRNA
である、請求項15記載の医薬。 - Cas9が化膿性レンサ球菌由来Cas9である、請求項16記載の医薬。
- ガイドRNAが、
(1)配列番号1もしくは配列番号2で示される核酸配列を含むキメラRNA、又は
(2)(i) 配列番号3もしくは配列番号4で示される核酸配列を含むcrRNA、および
(ii)配列番号7もしくは配列番号8で示される核酸配列を含むtracrRNA
である、請求項15記載の医薬。 - 筋ジストロフィーの予防・治療薬である、請求項15記載の医薬。
- 修復型ジストロフィンタンパク産生薬である、請求項15記載の医薬。
- 哺乳動物に対して請求項6記載の組成物の有効量を投与することを特徴とする、該哺乳動物における筋ジストロフィーの予防・治療方法。
- 哺乳動物に対して請求項6記載の組成物の有効量を投与することを特徴とする、該哺乳動物における修復型ジストロフィンタンパク産生方法。
- 筋ジストロフィーの予防・治療剤を製造するための、請求項6記載の組成物の使用。
- 筋ジストロフィーの予防・治療に使用するための、請求項6記載の組成物。
- 請求項1記載の組成物と細胞とを接触させる工程を含む、標的遺伝子座が改変された細胞の製造方法。
- (1)請求項1記載の組成物と非ヒト哺乳動物の受精卵、胚性幹細胞もしくは卵母細胞とを接触させる工程、
(2)標的遺伝子座が改変された前記受精卵、胚性幹細胞もしくは卵母細胞を選択する工程、及び
(3)前記選択された受精卵、胚性幹細胞もしくは卵母細胞を非ヒト哺乳動物の雌性動物に移植する工程
を含む、標的遺伝子座が改変された非ヒト哺乳動物の作製方法。 - 前記ガイドRNAが2種類以上のガイドRNAである、請求項27記載の製造方法。
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