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CRISPR-Cas9基因编辑技术治疗镰状细胞病和β地中海贫血
CRISPR-Cas9 Gene Editing for Sickle Cell Disease and β-Thalassemia


Haydar Frangoul ... 其他 • 2021.01.21
相关阅读
• β地中海贫血 • BCL11A基因沉默策略增加镰状细胞病患者的胎儿血红蛋白 • 通过BCL11A转录后基因沉默策略治疗镰状细胞病 • 输血依赖型β-地中海贫血患者的基因治疗 • 一类通过DNA编辑发挥作用的新型药物

述评一

CRISPR-Cas9基因编辑技术治疗β地中海贫血和镰状细胞贫血

——从基础研究向临床转化

 

张楹*,殷昊

武汉大学医学研究院;武汉大学教育部免疫与代谢前沿科学中心

*通讯作者

 

地中海贫血和镰状细胞贫血是由于珠蛋白单基因突变而引起血红蛋白生成障碍,是全球分布最广、影响人群最多的遗传病。全世界每年新增约6万输血依赖型地中海贫血患者和30万镰状细胞贫血患者。我国约有3,000万地中海贫血基因携带者,已有输血依赖型患者30多万。地中海贫血是我国影响最大、发病率最高的遗传病。只有不到20%的地中海贫血和镰状细胞贫血患者可找到合适的配型进行异体骨髓移植实现治愈,而大部分患者只能依靠常规输血等辅助性疗法,无法治愈。

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摘要


输血依赖型β地中海贫血(TDT)和镰状细胞病(SCD)是严重的单基因病,具有严重且可能危及生命的临床表现。BCL11A是可抑制红系细胞内γ珠蛋白表达和胎儿血红蛋白的转录因子。我们对来自健康供者的CD34+造血干细胞和祖细胞实施电穿孔,并应用成簇规律间隔短回文重复序列(CRISPR)-Cas9对BCL11A红系特异性增强子实施靶向编辑。该位点约80%的等位基因被修饰,且无脱靶编辑的证据。接受清髓性预处理后,2例患者(1例TDT患者,1例SCD患者)接受了靶向相同BCL11A增强子的CRISPR-Cas9编辑的自体CD34+细胞。1年多后,2例患者骨髓和血液内的等位基因编辑水平高,分布在全部细胞内的胎儿血红蛋白增加,摆脱输血依赖,并且(SCD患者)不再发生血管闭塞(由CRISPR Therapeutics和Vertex Pharmaceuticals资助,CLIMB THAL-111在ClinicalTrials.gov注册号为NCT03655678,CLIMB SCD-121注册号为NCT03745287)。

TDT和SCD是全球范围内最常见的单基因病,每年诊断出约6万例TDT患者和30万例SCD患者1-3。两种疾病均由血红蛋白β亚基基因(HBB)突变引起。引起TDT4HBB突变导致β珠蛋白合成减少(β+)或缺失(β0),以及血红蛋白的类α和类β珠蛋白(如β、γ和δ)链之间的不平衡,这会导致无效性红细胞生成5,6。镰刀状血红蛋白是HBB点突变(第6位氨基酸,谷氨酸被缬氨酸取代)的结果。脱氧镰刀状血红蛋白聚合会导致红细胞变形、溶血、贫血、引起疼痛的血管闭塞发作、不可逆的终末器官损伤和预期寿命缩短5

治疗方案主要包括:对TDT患者采取输血和铁螯合治疗7,对SCD患者采取疼痛管理、输血和羟基脲治疗8。最近批准的治疗药物(包括luspatercept 9和crizanlizumab 10)减少了TDT患者的输血需求,并且降低了SCD患者的血管闭塞发作率,但这些治疗都不能解决疾病的根本原因,也不能完全缓解疾病表现。异基因骨髓移植可治愈TDT和SCD,但在适合骨髓移植的患者中,只有不到20%有人类白细胞抗原相合的亲缘供者11-13。betibeglogene autotemcel是一种基于慢病毒载体的基因添加(gene-addition)制品,已被欧盟批准用于治疗携带非β0突变,且无相合同胞供者的TDT患者14,并且目前正在研究将其用于β00 TDT基因型患者和SCD患者15,16。此外,研究证明通过慢病毒编码的微RNA适应性短发夹RNA分子将BCL11A进行红系特异性敲减可重新激活γ珠蛋白基因,该疗法目前处于早期临床开发阶段17-19

胎儿血红蛋白(包括两个α链和两个γ链)水平升高与TDT和SCD患者发病率和死亡率降低相关20-22。胎儿血红蛋白的生成受到发育过程的调节,因此子宫内生成的γ珠蛋白的水平在产后降低,与此同时β珠蛋白和成人血红蛋白(由两个α链和两个β链组成)的生成增加。患TDT或SCD的新生儿和婴儿在胎儿血红蛋白仍保持较高水平期间通常无症状,并在1岁前随着胎儿血红蛋白合成的减少而出现症状23,24(图1A)。如果TDT或SCD患者同时患了遗传性胎儿血红蛋白持续存在症,即胎儿血红蛋白的表达持续至成人期,则这些患者极少发病或不发病25

 

1. CTX001分子学方法和临床前研究

图A显示胎儿血红蛋白(HbF)在婴儿出生后不久转变为成人血红蛋白(HbA),并显示转录因子BCL11A如何介导对γ珠蛋白(胎儿血红蛋白的一种成分)的抑制。如果患者无法生成有功能或足够的β珠蛋白,则在出生后约3个月时,会随着胎儿血红蛋白水平的下降出现症状。SCD表示镰状细胞病,TDT表示输血依赖型β地中海贫血。图B显示将CRISPR-Cas9引导至BCL11A红系特异性增强子区域的单指导RNA(SgRNA)的靶向编辑位点(sgRNA序列见补充附录)。5个BCL11A外显子被绘制为金色方框。GATA1表示GATA1转录因子的结合位点,PAM表示前间隔序列邻近基序(NGG,紧接在Cas9靶向DNA序列之后的特定DNA序列)。图C显示红系细胞编辑和分化后,胎儿血红蛋白的临床前数据(表示为占全部血红蛋白的百分比)。数据来自10名健康供者的样本。误差条表示标准差。图D显示脱靶情况评估结果。GUIDE-seq表示通过测序对双链断裂进行的全基因组无偏鉴定,HSPC表示造血干细胞和祖细胞。为了确定位点,对3个CD34+ HSPC健康供者样本分别进行一次GUIDE-seq。为了确认位点,然后对4个CD34+ HSPC健康供者样本进行杂交捕获。每项实验中的在靶等位基因编辑得到证实,平均值为57%,此外在通过GUIDE-seq和序列同源性确定的任何位点均未观察到可测的脱靶编辑。获得Canver和Orkin允许后,我们对图A进行了改编24

 

全基因组关联研究已发现与成人的胎儿血红蛋白表达增加相关的单核苷酸多态性(SNP)26。其中一些SNP位于2号染色体上的BCL11A位点,并与较低的TDT和SCD严重程度相关27。BCL11A是一种含锌指的转录因子,可抑制红系细胞中γ珠蛋白和胎儿血红蛋白的表达;与胎儿血红蛋白相关的SNP位于红系特异性增强子中,可下调BCL11A的表达,并增加胎儿血红蛋白的表达1,23

CRISPR-Cas9核酸酶系统是可切割噬菌体或质粒DNA的细菌免疫系统,因此通过其能够以可编程的方式靶向特定基因组DNA位点的插入或缺失(indel)28,29。为了重现遗传性胎儿血红蛋白持续存在症的表型,我们使用CRISPR-Cas9基因编辑技术对造血干细胞和祖细胞(HSPC)的BCL11A红系特异性的增强子区域进行处理,以减少红系细胞中的BCL11A表达,恢复γ珠蛋白合成,并重新激活胎儿血红蛋白的生成30,31(图1B)。

我们在本文中报告了2例患者(1例患TDT,另1例患SCD),他们接受了CTX001输入(经过CRISPR-Cas9编辑的自体CD34+ HSPC,以基因编辑方式重新激活了胎儿血红蛋白的生成),并分别被纳入CLIMB THAL-111(纳入TDT患者)和CLIMB SCD-121试验(纳入SCD患者)。





作者信息

Haydar Frangoul, M.D., David Altshuler, M.D., Ph.D., M. Domenica Cappellini, M.D., Yi-Shan Chen, Ph.D., Jennifer Domm, M.D., Brenda K. Eustace, Ph.D., Juergen Foell, M.D., Josu de la Fuente, M.D., Ph.D., Stephan Grupp, M.D., Ph.D., Rupert Handgretinger, M.D., Tony W. Ho, M.D., Antonis Kattamis, M.D., Andrew Kernytsky, Ph.D., Julie Lekstrom-Himes, M.D., Amanda M. Li, M.D., Franco Locatelli, M.D., Markus Y. Mapara, M.D., Ph.D., Mariane de Montalembert, M.D., Damiano Rondelli, M.D., Akshay Sharma, M.B., B.S., Sujit Sheth, M.D., Sandeep Soni, M.D., Martin H. Steinberg, M.D., Donna Wall, M.D., Angela Yen, Ph.D., and Selim Corbacioglu, M.D.
From the Sarah Cannon Center for Blood Cancer at the Children’s Hospital at TriStar Centennial, Nashville (H.F., J.D.), and St. Jude Children’s Research Hospital, Memphis (A.S.) — both in Tennessee; Vertex Pharmaceuticals (D.A., B.K.E., J.L.-H., A.Y.) and Boston University School of Medicine (M.H.S.), Boston, and CRISPR Therapeutics, Cambridge (Y.-S.C., T.W.H., A. Kernytsky, S. Soni) — both in Massachusetts; the University of Milan, Milan (M.D.C.), and Ospedale Pediatrico Bambino Gesù Rome, Sapienza, University of Rome, Rome (F.L.); the University of Regensburg, Regensburg (J. Foell, S.C.), and Children’s University Hospital, University of Tübingen, Tübingen (R.H.) — both in Germany; Imperial College Healthcare NHS Trust, St. Mary’s Hospital, London (J. de la Fuente); Children’s Hospital of Philadelphia, Perelman School of Medicine at the University of Pennsylvania, Philadelphia (S.G.); the University of Athens, Athens (A. Kattamis); BC Children’s Hospital, University of British Columbia, Vancouver (A.M.L.), and the Hospital for Sick Children–University of Toronto, Toronto (D.W.) — both in Canada; Columbia University (M.Y.M.) and the Joan and Sanford I. Weill Medical College of Cornell University (S. Sheth), New York; Necker–Enfants Malades Hospital, Assistance Publique–Hôpitaux de Paris, University of Paris, Paris (M.M.); and the University of Illinois at Chicago, Chicago (D.R.). Address reprint requests to Dr. Frangoul at the Sarah Cannon Center for Blood Cancer at the Children’s Hospital at TriStar Centennial, 330 23rd Ave. N., Suite 450, Nashville, TN 37203, or at haydar.frangoul@hcahealthcare.com; or to Dr. Corbacioglu at Children’s Hospital Regensburg, University of Regensburg, Franz-Josef Strauss Allee 11, 93053 Regensburg, Germany, or at selim.corbacioglu@mac.com.

 

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