JP2021500058A - 胎児ヘモグロビンの発現レベルを増加させる方法 - Google Patents
胎児ヘモグロビンの発現レベルを増加させる方法 Download PDFInfo
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Abstract
Description
本出願は、2017年10月27日に提出された中国特許出願2017110277086の優先権を享有することを主張する。当該中国特許出願の開示内容は、その全体が引用により本出願に取り込まれる。
1.ヒト造血幹細胞の2番染色体の60495219番目から60495336番目のBCL11Aのゲノム領域を遺伝子編集技術によって破壊することを含む、
ヒト造血幹細胞における胎児ヘモグロビン(HbF)の発現を増加させる方法。
2.前記遺伝子編集技術は、ジンクフィンガーヌクレアーゼに基づく遺伝子編集技術、TALEN遺伝子編集技術またはCRISPR/Cas遺伝子編集技術である、項1に記載の方法。
3.前記遺伝子編集技術がCRISPR/Cas9遺伝子編集技術である、項2に記載の方法。
4.前記BCL11Aゲノムの標的ヌクレオチド配列は、配列番号3〜配列番号25から選択されるいずれか一つの配列と相補的である、項1〜3のいずれか一項に記載の方法。
5.前記BCL11Aゲノムの編集を実現するように、配列番号3〜配列番号25から選択されるいずれか一つの配列を含むsgRNAを前記造血幹細胞に導入することを含む、項3または4に記載の方法。
6.前記sgRNAは、2’−O−メチル類縁体及び/またはヌクレオチド間3’チオにより修飾されたものである、項5に記載の方法。
7.前記化学修飾は、前記sgRNAの5’末端の最初の1つ、2つ、及び/または3つの塩基及び/または3’末端の最後の1つの塩基の2’−O−メチル類縁体による修飾である、項6に記載の方法。
8.前記sgRNAと、Cas9をコードするヌクレオチドとを一緒に前記造血幹細胞に導入する、項3〜7のいずれか一項に記載の方法。
9.前記sgRNAと、Cas9をコードするヌクレオチドとをエレクトロポレーションによって一緒に前記造血幹細胞に導入する、項8に記載の方法。
10.前記エレクトロポレーションの条件は、200〜600V、0.5〜2msである、項9に記載の方法。
12.前記sgRNAとCas9をコードするヌクレオチドとを一緒に前記造血幹細胞に導入する、項11に記載の方法。
13.sgRNAと、Cas9をコードするヌクレオチドとをエレクトロポレーションによって一緒に前記造血幹細胞に導入する、項12に記載の方法。
14.前記エレクトロポレーションの条件は、200〜600V、0.5〜2msである、項13に記載の方法。
15.項1〜14のいずれか一項に記載の方法によって得られた造血幹細胞。
16.胎児ヘモグロビン(HbF)の発現が遺伝子改変によって増加したヒト造血幹細胞であって、前記造血幹細胞の2番染色体の60495219番目から60495336番目のBCL11Aゲノム領域における一つ以上の部位が遺伝子編集技術によって破壊された、胎児ヘモグロビン(HbF)の発現が遺伝子改変によって増加したヒト造血幹細胞。
17.項15または16に記載の造血幹細胞を分化培養することにより得られた、成熟赤血球になる前の分化の異なる段階にある前駆細胞。
18.項15または16に記載の造血幹細胞を分化培養することにより得られた成熟赤血球。
19.胎児ヘモグロビン(HbF)の発現が遺伝子改変によって増加した成熟赤血球またはその前駆細胞を製造する方法であって、
(a)項1〜14のいずれか一項に記載の方法を使用して遺伝子改変された造血幹細胞を得ることと、
(b)造血幹細胞赤血球系増殖および分化培地を使用して、前記遺伝子改変された造血幹細胞に対して造血幹細胞の赤血球系増殖および分化を行うこととを含み、
前記造血幹細胞赤血球系増殖および分化培地は、基礎培地と、成長因子組成物とを含み、前記成長因子組成物は、幹細胞成長因子(SCF)、インターロイキン3(IL−3)、およびエリスロポエチン(EPO)を含む、方法。
20.赤血球系分化脱核培地を使用して造血幹細胞の赤血球分化および脱核を行うことをさらに含み、
前記赤血球分化脱核培地は、基礎培地、成長因子、およびプロゲステロン受容体とグルココルチコイド受容体の拮抗薬および/または阻害剤を含む、
項19に記載の方法。
23.項15または16に記載の造血幹細胞、若しくは項17または22に記載の前駆細胞、若しくは項18または22に記載の成熟赤血球を含む、組成物。
24.項15または16に記載の造血幹細胞、若しくは項17または22に記載の前駆細胞、若しくは項18または22に記載の成熟赤血球を含む、医療製品。
25.項15または16に記載の造血幹細胞、若しくは請求項17または22に記載の前駆細胞、若しくは項18または22に記載の成熟赤血球の、それの必要がある被験者の疾患の予防または治療における使用。
26.前記疾患が貧血疾患、出血性疾患、腫瘍、または予防または治療のために大量の輸血を必要とする他の疾患である、項25に記載の使用。
27.前記疾患はβ−サラセミアまたは鎌状赤血球貧血である、請求項26に記載の使用。
28.前記被験者はヒトである、請求項25〜27のいずれか一項に記載の使用。
29.被験者の疾患を予防または治療するための薬剤または医療製品の調製における、項15または16に記載の造血幹細胞、若しくは項17または22に記載の前駆細胞、若しくは項18または22に記載の成熟赤血球の使用。
30.前記疾患が貧血疾患、出血性疾患、腫瘍、または予防または治療のために大量の輸血を必要とする他の疾患である、項29に記載の使用。
32.前記被験者はヒトである、項29〜31のいずれか一項に記載の使用。
33.配列番号3〜配列番号25から選択される一つのヌクレオチド配列を含む、sgRNA構築体。
34.2’−O−メチル類縁体及び/またはヌクレオチド間3’チオ修飾を含む、項33に記載の構築体。
35.前記化学修飾は、配列番号3〜配列番号25から選択される一つのヌクレオチド配列の5’末端の最初の1つ、2つ、及び/または3つの塩基及び/または3’末端の最後の1つの塩基の2’−O−メチル類縁体による修飾である、項34に記載の構築体。
36.項33〜35のいずれか一項に記載の構築体を含むベクター、宿主細胞または製剤。
37.造血幹細胞の遺伝子編集における、項33〜35のいずれか一項に記載の構築体の使用。
38.項15または16に記載の造血幹細胞、項17または22に記載の前駆細胞、若しくは項18または22に記載の成熟赤血球を被験者に投与することを含む、被験者の貧血疾患、出血性疾患、腫瘍、または予防または治療のために大量の輸血を必要とする他の疾患を治療または予防する方法。
39.前記疾患はβ−サラセミアまたは鎌状赤血球貧血である、項38に記載の方法。
40.前記被験者はヒトである、項39に記載の方法。
41.項33〜35のいずれか一項に記載のsgRNAの構築体、または項36に記載のベクターを含む、被験者の貧血疾患、出血性疾患、腫瘍、または予防または治療のために大量の輸血を必要とする他の疾患を治療または予防するキット。
42.Cas9mRNAをさらに含む、項41に記載のキット。
a)磁気ビーズ選別により、ヒト臍帯血からCD34陽性HSPCsを分離すること;
b)本発明に記載のいずれかの方法を使用して前記CD34陽性HSPCsを遺伝子改変すること;
c)追加の成長因子を無血清培地(Serum−free medium(SFME))に添加し、5〜10日、例えば、5日、6日、7日、8日、9日、10日、増殖および分化した後、HSPCsを赤血球前駆細胞に分化させること(この段階の培地を、造血幹細胞赤血球系増殖および分化培地(HSPCs erythroid expansion and differentiatiоn medium;HEEDMと略記する)となづける);
d)追加の成長因子を無血清培地(Serum−free medium(SFME))に添加し、7日間分化した後、成熟した赤血球を得ること(この段階の培地を、造血幹細胞赤血球系分化脱核培地(HSPCs erythroid differentiatiоn enucleatiоn medium;HEDEMと略記する)となづける)。
1−1:K562細胞を使用してエレクトロポレーション条件を調べる
造血幹細胞の供給源が少なく、一回単離するコストが高いため、本実施例では、エレクトロポレーション条件を調べるためのモデル細胞株として、癌細胞株K562(ATCC社から購入、ウェブサイト:https://www.atcc.оrg)を選択した。
実験ロット一:5×105のK562細胞をエレクトロポレーションにより形質転換し、GFPmRNA(配列番号1に示す配列)の量は5μgであり、BTX830エレクトロポレーターを使用し、それぞれ250V 1ms、360V 1ms、400V 1ms、500V 1msの条件下で、4日後にGFP発現及び7−AAD発現をフローサイトメトリー解析実験により検出した。GFPはエレクトロポレーションの効率を表し、7−AADはエレクトロポレーション後の細胞の成長状態、すなわち生存能力(viability)を表す。
atgagtaaaggagaagaacttttcactggagttgtcccaattcttgttgaattagatggtgatgttaatgggcacaaattttctgtcagtggagagggtgaaggtgatgcaacatacggaaaacttacccttaaatttatttgcactactggaaaactacctgttccatggccaacacttgtcactactttctcttatggtgttcaatgcttttcaagatacccagatcatatgaaacggcatgactttttcaagagtgccatgcccgaaggttatgtacaggaaagaactatatttttcaaagatgacgggaactacaagacacgtgctgaagtcaagtttgaaggtgatacccttgttaatagaatcgagttaaaaggtattgattttaaagaagatggaaacattcttggacacaaattggaatacaactataactcacacaatgtatacatcatggcagacaaacaaaagaatggaatcaaagttaacttcaaaattagacacaacattgaagatggaagcgttcaactagcagaccattatcaacaaaatactccaattggcgatggccctgtccttttaccagacaaccattacctgtccacacaatctgccctttcgaaagatcccaacgaaaagagagaccacatggtccttcttgagtttgtaacagctgctgggattacacatggcatggatgaactatacaaatag
前のステップで効率および生存率が最も高い条件、すなわち、300V 1msを選択し、5×105の造血幹細胞(ALLCELLS(上海)社から購入、www.allcells.c)に上記GFPmRNAをエレクトロポレーションにより形質転換し、4日後にGFPとCD34の発現状況を検出した。結果を図4と図5に示す。
A、BCL11Aのエンハンサー58K部位(この標的58K部位の配列は配列番号2に示される)について、「CRISPR RGEN TOOLS」ソフトウェアを使用してBCL11A(+58)部位に対して複数のsgRNAを設計し、化学修飾されたsgRNAを合成した。情報は図6、7、8に示す。
ctgccagtcctcttctaccccacccacgcccccaccctaatcagaggccaaacccttcctggagcctgtgataaaagcaactgttagcttgcactagactagcttcaaagttgtattgaccctggtgtgttatgtctaagagtagatgcc
cacaggctccaggaagggtt
atcagaggccaaacccttcc
ctaacagttgcttttatcac
ttgcttttatcacaggctcc
ttttatcacaggctccagga
tttatcacaggctccaggaa
tgggtggggtagaagaggac
gggcgtgggtggggtagaag
ttagggtgggggcgtgggtg
attagggtgggggcgtgggt
gattagggtgggggcgtggg
tctgattagggtgggggcgt
ctctgattagggtgggggcg
cacgcccccaccctaatcag
ttggcctctgattagggtgg
tttggcctctgattagggtg
gtttggcctctgattagggt
ggtttggcctctgattaggg
aagggtttggcctctgatta
gaagggtttggcctctgatt
actcttagacataacacacc
cttcaaagttgtattgaccc
ctcttagacataacacacca
リバースプライマー:gggaagctccaaactctcaa(配列番号30)
gacaagaagtacagcatcggcctggacatcggcaccaactctgtgggctgggccgtgatcaccgacgagtacaaggtgcccagcaagaaattcaaggtgctgggcaacaccgaccggcacagcatcaagaagaacctgatcggagccctgctgttcgacagcggcgaaacagccgaggccacccggctgaagagaaccgccagaagaagatacaccagacggaagaaccggatctgctatctgcaagagatcttcagcaacgagatggccaaggtggacgacagcttcttccacagactggaagagtccttcctggtggaagaggataagaagcacgagcggcaccccatcttcggcaacatcgtggacgaggtggcctaccacgagaagtaccccaccatctaccacctgagaaagaaactggtggacagcaccgacaaggccgacctgcggctgatctatctggccctggcccacatgatcaagttccggggccacttcctgatcgagggcgacctgaaccccgacaacagcgacgtggacaagctgttcatccagctggtgcagacctacaaccagctgttcgaggaaaaccccatcaacgccagcggcgtggacgccaaggccatcctgtctgccagactgagcaagagcagacggctggaaaatctgatcgcccagctgcccggcgagaagaagaatggcctgttcggcaacctgattgccctgagcctgggcctgacccccaacttcaagagcaacttcgacctggccgaggatgccaaactgcagctgagcaaggacacctacgacgacgacctggacaacctgctggcccagatcggcgaccagtacgccgacctgtttctggccgccaagaacctgtccgacgccatcctgctgagcgacatcctgagagtgaacaccgagatcaccaaggcccccctgagcgcctctatgatcaagagatacgacgagcaccaccaggacctgaccctgctgaaagctctcgtgcggcagcagctgcctgagaagtacaaagagattttcttcgaccagagcaagaacggctacgccggctacattgacggcggagccagccaggaagagttctacaagttcatcaagcccatcctggaaaagatggacggcaccgaggaactgctcgtgaagctgaacagagaggacctgctgcggaagcagcggaccttcgacaacggcagcatcccccaccagatccacctgggagagctgcacgccattctgcggcggcaggaagatttttacccattcctgaaggacaaccgggaaaagatcgagaagatcctgaccttccgcatcccctactacgtgggccctctggccaggggaaacagcagattcgcctggatgaccagaaagagcgaggaaaccatcaccccctggaacttcgaggaagtggtggacaagggcgcttccgcccagagcttcatcgagcggatgaccaacttcgataagaacctgcccaacgagaaggtgctgcccaagcacagcctgctgtacgagtacttcaccgtgtataacgagctgaccaaagtgaaatacgtgaccgagggaatgagaaagcccgccttcctgagcggcgagcagaaaaaggccatcgtggacctgctgttcaagaccaaccggaaagtgaccgtgaagcagctgaaagaggactacttcaagaaaatcgagtgcttcgactccgtggaaatctccggcgtggaagatcggttcaacgcctccctgggcacataccacgatctgctgaaaattatcaaggacaaggacttcctggacaatgaggaaaacgaggacattctggaagatatcgtgctgaccctgacactgtttgaggacagagagatgatcgaggaacggctgaaaacctatgcccacctgttcgacgacaaagtgatgaagcagctgaagcggcggagatacaccggctggggcaggctgagccggaagctgatcaacggcatccgggacaagcagtccggcaagacaatcctggatttcctgaagtccgacggcttcgccaacagaaacttcatgcagctgatccacgacgacagcctgacctttaaagaggacatccagaaagcccaggtgtccggccagggcgatagcctgcacgagcacattgccaatctggccggcagccccgccattaagaagggcatcctgcagacagtgaaggtggtggacgagctcgtgaaagtgatgggccggcacaagcccgagaacatcgtgatcgaaatggccagagagaaccagaccacccagaagggacagaagaacagccgcgagagaatgaagcggatcgaagagggcatcaaagagctgggcagccagatcctgaaagaacaccccgtggaaaacacccagctgcagaacgagaagctgtacctgtactacctgcagaatgggcgggatatgtacgtggaccaggaactggacatcaaccggctgtccgactacgatgtggaccatatcgtgcctcagagctttctgaaggacgactccatcgacaacaaggtgctgaccagaagcgacaagaaccggggcaagagcgacaacgtgccctccgaagaggtcgtgaagaagatgaagaactactggcggcagctgctgaacgccaagctgattacccagagaaagttcgacaatctgaccaaggccgagagaggcggcctgagcgaactggataaggccggcttcatcaagagacagctggtggaaacccggcagatcacaaagcacgtggcacagatcctggactcccggatgaacactaagtacgacgagaatgacaagctgatccgggaagtgaaagtgatcaccctgaagtccaagctggtgtccgatttccggaaggatttccagttttacaaagtgcgcgagatcaacaactaccaccacgcccacgacgcctacctgaacgccgtcgtgggaaccgccctgatcaaaaagtaccctaagctggaaagcgagttcgtgtacggcgactacaaggtgtacgacgtgcggaagatgatcgccaagagcgagcaggaaatcggcaaggctaccgccaagtacttcttctacagcaacatcatgaactttttcaagaccgagattaccctggccaacggcgagatccggaagcggcctctgatcgagacaaacggcgaaaccggggagatcgtgtgggataagggccgggattttgccaccgtgcggaaagtgctgagcatgccccaagtgaatatcgtgaaaaagaccgaggtgcagacaggcggcttcagcaaagagtctatcctgcccaagaggaacagcgataagctgatcgccagaaagaaggactgggaccctaagaagtacggcggcttcgacagccccaccgtggcctattctgtgctggtggtggccaaagtggaaaagggcaagtccaagaaactgaagagtgtgaaagagctgctggggatcaccatcatggaaagaagcagcttcgagaagaatcccatcgactttctggaagccaagggctacaaagaagtgaaaaaggacctgatcatcaagctgcctaagtactccctgttcgagctggaaaacggccggaagagaatgctggcctctgccggcgaactgcagaagggaaacgaactggccctgccctccaaatatgtgaacttcctgtacctggccagccactatgagaagctgaagggctcccccgaggataatgagcagaaacagctgtttgtggaacagcacaagcactacctggacgagatcatcgagcagatcagcgagttctccaagagagtgatcctggccgacgctaatctggacaaagtgctgtccgcctacaacaagcaccgggataagcccatcagagagcaggccgagaatatcatccacctgtttaccctgaccaatctgggagcccctgccgccttcaagtactttgacaccaccatcgaccggaagaggtacaccagcaccaaagaggtgctggacgccaccctgatccaccagagcatcaccggcctgtacgagacacggatcgacctgtctcagctgggaggcgac
cacctcagcagaaacaaagttatc
gggaagctccaaactctcaa
本実験は、遺伝子編集された臍帯血由来の造血幹細胞のコロニー形成単位(CFU、colony−formation units)の検出に係る。
300V 1msのエレクトロポレーション条件を選択し、Cas9mRNAおよびEnhancer−2をエレクトロポレーションにより臍帯血由来の造血幹細胞に導入し、800〜1000の細胞を1ml H4434(カナダSTEM CELLS TECHNOLOGIESから購入)とIMDM(Thermo Fisherから購入)およびFBS(Thermo Fisherから購入)の混合液に再懸濁し、14日後にCFU−M、BFU−E、CFU−E、CFU−G、CFU−GM、GEMMなど形成された異なる形のコロニー数を顕微鏡で観察し、その結果を図11に示す。図11は、Cas9 mRNAとBCL11Aのエンハンサー‐2 sgRNAをエレクトロポレーションにより臍帯血由来のCD34陽性造血幹細胞に導入してから2日後、インビトロコロニー形成実験(CFU検出)を実施し、14日後に異なる血液系のコロニー数をカウントした結果を示す図である。BFU−E、CFU−M、CFU−GM、CFU−E、CFU−G、CFU−MMは、赤血球系、骨髄系、リンパ系などの血液系の異なる系統のコロニー形成を表す。その中で、Mockは遺伝子編集されていない細胞を表す。
300V 1msのエレクトロポレーション条件を選択し、Cas9mRNAおよびEnhancer−2をエレクトロポレーションにより臍帯血由来の造血幹細胞に導入し、放射計で照射されたNPG免疫不全マウスモデル(北京維通達生物技術有限公司(Beijing Vitalstar Biotechnology,Inc.)から購入)に移植した。移植して6週間、8週間、10週間、12週間、16週間後、末梢血でヒトCD45陽性細胞とマウスCD45の発現を検出すると共に、移植して16週間後の骨髄と脾臓でヒトCD45とマウスCD45の発現を検出し、その結果は図12と図14に示す。マウスへの移植方法は次の通りである。細胞移植の24時間前に、1.0Gyの照射を行ってマウスモデルの骨髄を除去した。そして、20μL 0.9%生理食塩水で再懸濁した1.0×106の細胞をマウスの尾静脈に注射し、クリーングレードの動物舎に飼育した。
4−1.赤血球の分化
300V 1msのエレクトロポレーション条件を選択し、Cas9mRNAおよびEnhancer−2をエレクトロポレーションにより臍帯血由来の造血幹細胞に導入し、次の「二段階法」分化スキームを使用して分化させた。
上記の赤血球が分化した細胞のmRNAを抽出し、cDNAに逆転写し、BCL11A、HBB、HBGなどの遺伝子の発現を蛍光定量PCRによって検出し、その結果を図18に示す。その結果、BCL11Aのエンハンサー部位の遺伝子がノックアウトされた細胞のBCL11A遺伝子発現が1倍ダウンレギュレーションされ、HBG発現が1倍と増加した。
5−1.β−サラセミア患者の末梢血からの造血幹細胞の単離
従来の磁気ビーズ選別によってCD34陽性の造血幹細胞を得た。結果を図20に示す。
300V 1msのエレクトロポレーション条件を選択し、Cas9 mRNAとEnhancer−2、Enhancer−3、Enhancer−4、Enhancer−5およびEnhancer−6をエレクトロポレーションにより、β−サラセミア患者の末梢血からの造血幹細胞に導入し、4日後、Indels効率を検出し、その結果を図21に示す。3名の異なる患者の末梢血からの造血幹細胞において、5つのsgRNAは何れも効率的な遺伝子編集を実現でき、その中で、Enhancer−2の効率が最も高く、70%以上にも達していることは示されている。
実施例5で遺伝子編集された造血幹細胞をインビトロで赤血球系分化させ、その方法は実施例4−1を参照できる。分化の結果を図22に示す。実験の結果、対照群、Enhancer−2、Enhancer−3、Enhancer−4、Enhancer−5およびEnhancer−6の赤血球系分化効率が互いに近く、いずれもCD71とCD235a細胞膜タンパク質を高く発現したことは示されている。
300V 1msのエレクトロポレーション条件を選択し、Cas9mRNAおよびEnhancer−2をエレクトロポレーションにより貧血患者の末梢血由来の造血幹細胞に導入し、500〜800の細胞を1ml H4434(カナダSTEM CELLS TECHNOLOGIESから購入)とIMDM(Thermo Fisherから購入)およびFBS(Thermo Fisherから購入)の混合液に再懸濁し、14日後にCFU−M、BFU−E、CFU−E、CFU−G、CFU−GM、GEMMなど形成された異なる形のコロニー数を顕微鏡で観察し、その結果を図25に示す。
本実験は遺伝子シーケンシング法に係り、具体的には、遺伝子編集された貧血患者の末梢血由来の造血幹細胞のオフターゲット効果(оff−target effect)を次世代シーケンシング技術(Next generation sequencing,NGS)によって分析する。
Claims (39)
- ヒト造血幹細胞の2番染色体の60495219番目から60495336番目のBCL11Aのゲノム領域を遺伝子編集技術によって破壊することを含む、
ヒト造血幹細胞における胎児ヘモグロビン(HbF)の発現を増加させる方法。 - 前記遺伝子編集技術は、ジンクフィンガーヌクレアーゼに基づく遺伝子編集技術、TALEN遺伝子編集技術またはCRISPR/Cas遺伝子編集技術である、請求項1に記載の方法。
- 前記遺伝子編集技術がCRISPR/Cas9遺伝子編集技術である、請求項2に記載の方法。
- 前記BCL11Aゲノムの標的ヌクレオチド配列は、配列番号3〜配列番号25から選択されるいずれか一つの配列と相補的である、請求項1〜3のいずれか一項に記載の方法。
- 前記BCL11Aゲノムの編集を実現するように、配列番号3〜配列番号25から選択されるいずれか一つの配列を含むsgRNAを前記造血幹細胞に導入することを含む、請求項3または4に記載の方法。
- 前記sgRNAは、2’−O−メチル類縁体及び/またはヌクレオチド間3’チオにより修飾されたものである、請求項5に記載の方法。
- 前記化学修飾は、前記sgRNAの5’末端の最初の1つ、2つ、及び/または3つの塩基及び/または3’末端の最後の1つの塩基の2’−O−メチル類縁体による修飾である、請求項6に記載の方法。
- 前記sgRNAと、Cas9をコードするヌクレオチドとを一緒に前記造血幹細胞に導入する、請求項3〜7のいずれか一項に記載の方法。
- 前記sgRNAと、Cas9をコードするヌクレオチドとをエレクトロポレーションによって一緒に前記造血幹細胞に導入する、請求項8に記載の方法。
- 前記エレクトロポレーション条件は、200〜600V、0.5〜2msである、請求項9に記載の方法。
- 配列番号3〜配列番号25から選択されるいずれか一つの配列を含む、2’−O−メチル類縁体及び/またはヌクレオチド間3’チオにより修飾されたsgRNAを造血幹細胞に導入することを含む、CRISPR/Cas9システムを介してインビトロで造血幹細胞を効率的に編集する方法。
- 前記sgRNAとCas9をコードするヌクレオチドとを一緒に前記造血幹細胞に導入する、請求項11に記載の方法。
- sgRNAと、Cas9をコードするヌクレオチドとをエレクトロポレーションによって一緒に前記造血幹細胞に導入する、請求項12に記載の方法。
- 前記エレクトロポレーション条件は、200〜600V、0.5〜2msである、請求項13に記載の方法。
- 請求項1〜14のいずれか一項に記載の方法によって得られた造血幹細胞。
- 胎児ヘモグロビン(HbF)の発現が遺伝子改変によって増加したヒト造血幹細胞であって、前記造血幹細胞の2番染色体の60495219番目から60495336番目のBCL11Aゲノム領域が遺伝子編集技術によって破壊された、ヒト造血幹細胞。
- 請求項15または16に記載の造血幹細胞を分化培養することにより得られた、成熟赤血球の前の分化の異なる段階にある前駆細胞。
- 請求項15または16に記載の造血幹細胞を分化培養することにより得られた成熟赤血球。
- 胎児ヘモグロビン(HbF)の発現が遺伝子改変によって増加した成熟赤血球またはその前駆細胞を製造する方法であって、
(a)請求項1〜14のいずれか一項に記載の方法を使用して遺伝子改変された造血幹細胞を得ることと、
(b)造血幹細胞赤血球系増殖および分化培地を使用して、前記遺伝子改変された造血幹細胞に対して造血幹細胞赤血球系増殖および分化を行うこととを含み、
前記造血幹細胞赤血球系増殖および分化培地は、基礎培地と、成長因子組成物とを含み、前記成長因子組成物は、幹細胞成長因子(SCF)、インターロイキン3(IL−3)、およびエリスロポエチン(EPO)を含む、方法。 - 赤血球系分化脱核培地を使用して造血幹細胞の赤血球系分化および脱核を行うことをさらに含み、
前記赤血球分化脱核培地は、基礎培地、成長因子、およびプロゲステロン受容体とグルココルチコイド受容体の拮抗薬および/または阻害剤を含む、
請求項19に記載の方法。 - 前記赤血球系分化脱核培地における成長因子はエリスロポエチン(EPO)を含み、前記プロゲステロン受容体とグルココルチコイド受容体の拮抗薬および/または阻害剤は、下記化合物(I)〜(IV)から選択されるいずれか1つまたは2つ以上である、請求項20に記載の方法。
- 請求項15または16に記載の造血幹細胞、若しくは請求項17に記載の前駆細胞、若しくは請求項18に記載の成熟赤血球を含む、組成物。
- 請求項15または16に記載の造血幹細胞、若しくは請求項17に記載の前駆細胞、若しくは請求項18に記載の成熟赤血球を含む、医療製品。
- 請求項15または16に記載の造血幹細胞、若しくは請求項17に記載の前駆細胞、若しくは請求項18に記載の成熟赤血球のそれの必要がある被験者の疾患の予防または治療における、使用。
- 前記疾患が貧血疾患、出血性疾患、腫瘍、または予防または治療のために大量の輸血を必要とする他の疾患である、請求項24に記載の使用。
- 前記疾患はβ−サラセミアまたは鎌状赤血球貧血である、請求項25に記載の使用。
- 前記被験者はヒトである、請求項24〜26のいずれか一項に記載の使用。
- 被験者の疾患を予防または治療するための薬剤または医療製品の調製における、請求項15または16に記載の造血幹細胞、若しくは請求項17に記載の前駆細胞、若しくは請求項18に記載の成熟赤血球の使用。
- 前記疾患が貧血疾患、出血性疾患、腫瘍、または予防または治療のために大量の輸血を必要とする他の疾患である、請求項28に記載の使用。
- 前記疾患はβ−サラセミアまたは鎌状赤血球貧血である、請求項29に記載の使用。
- 前記被験者はヒトである、請求項28〜30のいずれか一項に記載の使用。
- 配列番号3〜配列番号25から選択される一つのヌクレオチド配列を含む、sgRNA構築体。
- 2’−O−メチル類縁体及び/またはヌクレオチド間3’チオ修飾を含む、請求項32に記載の構築体。
- 前記化学修飾は、配列番号3〜配列番号25から選択される一つのヌクレオチド配列の5’末端の最初の1つ、2つ、及び/または3つの塩基及び/または3’末端の最後の1つの塩基の2’−O−メチル類縁体による修飾である、請求項33に記載の構築体。
- 請求項32〜34のいずれか一項に記載の構築体を含むベクター、宿主細胞または製剤。
- 造血幹細胞の遺伝子編集における、請求項32〜34のいずれか一項に記載の構築体の使用。
- 請求項15または16に記載の造血幹細胞、請求項17に記載の前駆細胞、若しくは請求項18に記載の成熟赤血球を被験者に投与することを含む、被験者の貧血疾患、出血性疾患、腫瘍、または予防または治療のために大量の輸血を必要とする他の疾患を治療または予防する方法。
- 前記疾患はβ−サラセミアまたは鎌状赤血球貧血である、請求項37に記載の方法。
- 前記被験者はヒトである、請求項38に記載の方法。
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CN112011576A (zh) * | 2019-05-31 | 2020-12-01 | 华东师范大学 | Crispr基因编辑技术在治疗地中海贫血中的应用 |
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CN110511909B (zh) * | 2019-07-29 | 2022-01-04 | 吉林大学 | 体外扩增造血干细胞的生长因子组合物及其应用 |
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CN112442516A (zh) * | 2019-08-29 | 2021-03-05 | 广州辑因医疗科技有限公司 | 一种高效修复环状铁粒幼细胞性贫血基因突变的方法 |
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