,1, 洪涛1, 秦鑫2, 梁英民3, 张萍,1Recent advance on genome editing for therapy of β-hemoglobinopathies
Jiawei Liu,1, Tao Hong1, Xin Qin2, Yingmin Liang3, Ping Zhang,1编委: 徐湘民
收稿日期:2017-09-28修回日期:2017-12-18网络出版日期:2018-01-09
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Received:2017-09-28Revised:2017-12-18Online:2018-01-09
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作者简介 About authors
作者简介:刘佳伟,本科,专业方向：生物技术E-mail:
通讯作者:张萍,博士,副教授,研究方向：遗传病的分子机制E-mail:
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刘佳伟, 洪涛, 秦鑫, 梁英民, 张萍. β-血红蛋白病基因组编辑治疗的研究进展. 遗传[J], 2018, 40(2): 95-103 doi:10.16288/j.yczz.17-215
Jiawei Liu, Tao Hong, Xin Qin, Yingmin Liang, Ping Zhang.
β-血红蛋白病(β-hemoglobinopathies)包括镰状红细胞贫血症和β-地中海贫血症两种类型,均是由β珠蛋白的突变所导致的疾病,为全世界最常见的单基因遗传病[1,2]。镰状红细胞贫血症产生的原因是β珠蛋白基因的第6外显子由GTG突变为GAG,从而导致β-珠蛋白第6位氨基酸谷氨酸(Glu)被缬氨酸(Lys)所代替,使得正常的血红蛋白(haemoglobin A,HbA)被镰状血红蛋白(haemoglobin sickle, HbS)所取代。而β-地中海贫血症则由β珠蛋白基因内多样化的点突变或者该基因部分片段缺失所造成。在临床上,目前β-血红蛋白病多以支持性治疗为主,如镰状红细胞贫血病常采用控制疼痛,促进水合和服用羟基脲等方法进行治疗,而β-地中海贫血则以输血或者铁螯合疗法为主要治疗方法[3]。虽然异基因骨髓移植是治愈性方案,但供体来源的匮乏和移植物抗宿主病等并发症大大限制了其临床应用,迫切需要探索新的治愈方案[4]。
人类血红蛋白由2条α珠蛋白链和2条β珠蛋白链所组成。前者由位于第16号染色体上的α珠蛋白基因座上的3个基因(§、α1和α2)中任一个所编码,而后者由位于第11号染色体上的β珠蛋白基因座的5个基因(ε、Gγ、Aγ、δ和β)中的任一个所编码。珠蛋白相关基因的表达在个体发育过程中均受到严格的调控[4]。在胚胎早期,β珠蛋白基因座上的ε、γ 珠蛋白基因分别在造血组织卵黄囊及造血肝组织中高表达。随着胎儿的发育成熟,ε、γ 珠蛋白基因表达逐渐沉默, β珠蛋白表达逐渐增加,由血红蛋白A 逐渐替代胎儿血红蛋白(human fetal hemoglobin, HbF)。胎儿血红蛋白HbF的表达在出生后数月内被关闭,越来越多的证据表明在患者中重新诱导HbF的表达可以有效治疗β-地中海贫血症[5]。对该病的遗传分子机制的深入认识催生了基因治疗的尝试。
β-血红蛋白病基因治疗的主要策略是基因补足,即在患者造血干祖细胞中过表达正常的β珠蛋白或者γ珠蛋白继而回输给患者。目前已有多个慢病毒表达系统推向临床研究,但是临床试验结果呈现以下问题：(1)导入的载有正确基因的整合型载体会一直存在于病人基因组中,安全性存在极大隐患; (2)导入的正常基因的表达受控于载体上的启动子以及调控元件,表达量往往不够以及表达水平不受原体内元件的精密调控,无法改善重症患者的临床症状;(3)导入的正常血红蛋白链和患者原有的异常血红蛋白链结合在一起,形成嵌合体,无法完全行使正常血红蛋白链的功能[6,7]。因而近20年过去了,始终没有产生具有临床推广价值的新疗法。
以锌指核酸酶(zinc finger nucleases, ZFNs)、转录激活型核酸酶(transcription activator like effector nucleases, TALENs)和CRISPR/Cas9(clustered regularly interspaced short palindromic repeats/CRISPR- associated protein 9, CRISPR/Cas9)等为代表的基因组编辑技术为这类疾病的治疗重新指明了方向[8,9,10]。这些技术可以通过造成DNA双链断裂切口,然后利用同源重组机制(homology-directed repair, HDR)或者非同源末端连接机制(nonhomologous end joining,NHEJ)方式修复,达到基因组编辑的目的。利用细胞内同源重组机制的策略为基因纠正方案,即通过提供一个染色体外的DNA修复模板,将模板和核酸酶共同导入靶细胞,利用DNA损伤修复后的同源重组机制修复靶细胞基因组中的DNA突变。利用非同源末端连接机制的策略称为基因破坏方案,可利用非同源末端连接途径产生基因组局部的插入或者缺失。若同时引入两个工程化的核酸酶则可实现靶序列的删除、倒位、重复,或者易位等染色体的重排[11]。目前以锌指核酸酶ZFN为编辑工具的方案已进入临床实验,如在HIV病人中以ZFN破坏其CD4+ T细胞的HIV病毒的辅受体Ccr5,可使绝大多数病人血液中HIV的滴度降低[12,13]。这一领域的进展非常迅速[9,14,15],利用这些核酸酶系统治疗β-血红蛋白病的研究也不断涌现。本文将将对β-血红蛋白病在基因组编辑研究和治疗方面的进展展开综述,以期为β-血红蛋白病新型治愈方案的研究以及基因组编辑技术的临床转化提供参考。
1 β-血红蛋白病的基因组编辑策略及 尝试
1.1 利用同源重组机制纠正β-血红蛋白病的基因缺陷
此治疗策略是将核酸酶和外源性的DNA修复模板同时导入到靶细胞内,利用核酸酶在靶序列造成DNA双链断裂切口,随后利用体内的同源重组机制进行DNA损伤修复以纠正患者的基因缺陷。相比传统的通过整合型表达载体导入正常的珠蛋白基因,这种方式可以避免由于随机整合导致的靶细胞基因组的不稳定性问题,并实现个性化的无痕修复。在镰状红细胞贫血疾病的基因组编辑治疗尝试方面,Hoban等[16,17]利用整合缺陷的慢病毒载体或电穿孔方法将DNA修复模板和靶向β-珠蛋白的ZFN核酸酶导入正常人CD34+造血干/祖细胞或者从镰状红细胞贫血患者分离的骨髓细胞中,发现对镰状红细胞贫血病变等位基因的体外修复效率可达20%。然而在免疫缺陷受体小鼠中,HDR修复的水平相比体外细胞实验降低了10~20倍[17]。DeWitt等[18]将由Cas9蛋白和未修饰的sgRNA偶联在一起组成Cas9 RNP (Cas9 ribonucleinprotein, Cas9 RNP)复合物,继而将该复合物和DNA修复模板一同导入人CD34+造血干祖细胞进行编辑。当编辑成功的人造血干祖细胞分化成为红细胞祖细胞时,正常血红蛋白的表达量增加。随后将编辑后细胞移植入免疫缺陷小鼠,修复后的镰状红细胞贫血基因的表达在所观察的16周内都是正常的。这些结果提示ZFN和CRISPR/ Cas9系统均可对镰状红细胞贫血病人造血干祖细胞进行成功的基因组编辑。在β-地中海贫血症的基因组编辑治疗尝试方面,Βahal等[19]利用PNAs (triplex-forming peptide nucleic acids)诱导DNA修复和催化基因组编辑,其中PNAs由中性的短肽骨架和若干核苷酸碱基所组成,可通过单链侵入方式诱导细胞通过核苷酸切除修复或者同源重组方式进行基因编辑。作为第三代的PNAs,γPNAs在PNAs的γ位置进行聚乙烯乙二醇的替代,从而大大提高了其结合靶序列DNA的能力和基因编辑效率。他们将γPNAs和供体DNA模板导入人CD34+造血干祖细胞,同时通过给予干细胞生长因子SCF(stem cell factor )激活c-Kit信号通路,可以在人造血干细胞中产生高水平的基因编辑。随后他们给β-地中海贫血模型小鼠注射SCF和包含γPNAs和供体DNA的纳米微粒,发现可以延缓疾病的症状,具体表现为血红蛋白水平持续增加到正常水平,网状细胞增多,脾肿大得以减轻[19]。进一步的检测表明7%的人造血干细胞中β-珠蛋白基因得到纠正,而且脱靶效应极低。这项研究将纳米传送系统和下一代γPNAs和SCF结合,实现了以最小的侵入性方法治疗血液系统遗传性疾病的目的[19]。然而由于HDR的频率相对比较低,其临床推广应用受到极大的限制。Dever 等[20]的工作带来了新的突破,他们将Cas9 RNP复合物与表达供体DNA模板及标签蛋白tNGFR (truncated neuron growth factor receptor,NGFR的胞内段被去除)的腺相关病毒载体共同导入到造血干祖细胞中,利用标签蛋白的抗体对编辑成功的造血干祖细胞进行富集,将实现靶向整合的细胞的比率从3.5%提高到90%以上。这一研究显示HDR介导的β-珠蛋白突变基因修复的低频率可以通过移植前对编辑成功的细胞进行筛选而克服。这些基因组编辑体系在镰状红细胞贫血治疗中的长期效果还需要进行进一步的评估。在突变类型复杂多样的β-地中海贫血治疗中应用这种策略时,需针对每一种突变制定对应的方案,这在临床转化上将是很大的挑战。1.2 利用非同源末端连接机制重新激活胎儿血红蛋白的表达
临床实践发现增加胎儿血红蛋白HbF水平可以缓解镰状红细胞贫血和β-地中海贫血症状。在设计基因组编辑方案时,需首先根据对血红蛋白表达调控的分子机制的充分认识,确定可编辑的靶基因位点,这些靶分子一般可抑制胎儿血红蛋白的表达,随后利用非同源末端连接机制关闭其表达,从而重新激活胎儿血红蛋白HbF的表达[5]。重新诱导的胎儿血红蛋白HbF可通过不同方式发挥作用,在镰状红细胞贫血病人中,足量的HbF可防止异常镰状形式的HbS多聚化。而在β-地中海贫血患者中,则可替代缺失的β-珠蛋白,减轻珠蛋白不同链的不平衡[4]。在实际应用中,一方面非同源末端连接的发生概率高于同源重组修复的发生概率,另一方面在基因组编辑方案的设计上,与利用同源重组修复机制需要针对每一病人的特定突变制定专一性的基因组编辑策略不同,对于不同的β-血红蛋白病人,可采用同一方案破坏某一分子的表达从而诱导出胎儿血红蛋白的表达即可,因而,这种策略相比利用同源重组机制纠正β-血红蛋白病的基因缺陷的策略更具有“one size for all”的优势。1.2.1 模拟珠蛋白基因的先天突变重新激活胎儿血红蛋白的表达
对多个β-血红蛋白病家系的遗传研究发现一些β珠蛋白基因座位存在罕见的有益突变,这些突变可导致可遗传的胎儿血红蛋白的持续表达从而缓解β-血红蛋白病患者的症状[5]。最初发现的是β珠蛋白基因簇大片段的缺失,其中一段包含约3.5 kb的γ珠蛋白沉默子区域。此外Aγ或Gγ珠蛋白基因启动子区的点突变和小的缺失也可使胎儿血红蛋白的表达持续到成年阶段[5]。新兴的基因编辑技术,使得在β-血红蛋白病人中模拟这些有益突变成为可能。Wienert等[21]在K562细胞中使用TALENs在Aγ启动子-175位置诱导了T→C的突变,形成了一个遗传性持续表达胎儿血红蛋白HPFH(hereditary persistance of fetal haemoglobin)的突变体。Traxler等[22]利用Cas9核酸酶在CD34+人造血干祖细胞中制造了γ 珠蛋白基因启动子区小的缺失,也实现了胎儿血红蛋白的持续性表达。携带这类先天性突变的β-血红蛋白病患者临床症状较轻表明这一策略具有长期有效性,在未来的临床治疗和应用方面有很大发展空间。
1.2.2 靶向调控胎儿血红蛋白的表达的重要因子
1.2.2.1 BCL11A或BCL11A增强子
BCL11A(B-cell lymphoma/leukemia 11A)是胎儿血红蛋白HbF表达的主要抑制因子[5],下调该因子的表达可以重新激活胎儿血红蛋白的表达。BCL11A具有一个12 kb的红系细胞特异性的增强子。在小鼠和人的红细胞系中剔除这段区域,可导致BCL11A在红细胞中的表达完全消失,而在B细胞系中剔除同一区域则不影响BCL11A在B细胞中的表达[23]。进一步的截短突变实验将该增强子的核心区域定位于一段20 bp的区域,此区域内含有红系特异转录因子GATA1的结合序列。对此段功能区域进行基因编辑设计是较有应用前景的治疗方案[24]。Chang等[25]在人骨髓来源CD34+细胞中以ZFN核酸酶破坏了此红系特异性启动子内的GATAA 基序,发现该方案可以将胎儿血红蛋白的表达水平上调到可以抑制异常HbS多聚化的水平。相比直接破坏BCL11A第2外显子的方案,这种方案具有以下优点：一是不影响红细胞成熟中的去核过程,二是可获得稳健的干祖细胞长期植入率,从而显著增加受体小鼠红细胞中胎儿血红蛋白的表达。另外利用siRNA技术在红细胞中特异性地调低BCL11A的表达也是目前正在研发的基因治疗方案[26]。
1.2.2.2 靶向LRF等其他因子
LRF(leukaemia/lymphoma related factor)是另一个重要的γ珠蛋白的抑制因子[27]。在红系细胞中以CRISPR/Cas9技术剔除LRF能导致γ珠蛋白的剧烈上调,其作用不依赖于BCL11A[27]。在CD34+人造血干祖细胞中敲除LRF导致红系发育的轻微延缓并同时诱导出γ珠蛋白的表达。虽然这一分子在调节γ珠蛋白上具有明显的作用,但是它在多种造血细胞分化中的作用尚不明确限制了它的临床应用。同样的问题也出现在靶向EHMT1/EHMT2(euchromatic histone methyltransferase1/2)和LIN28b通路的方案上,这些分子均可调节γ珠蛋白的表达,但因对其他发育环节的影响不明而进展缓慢[28,29]。此外,还有治疗方案靶向其他分子如广泛表达的KLF1(krüppel-like family of transcription factors)和MYΒ(myeloblastosis)[30,31],但因它们产生的副作用过大而无法进入临床应用。
2 临床转化平台的要素分析
2.1 靶细胞及造血干细胞的动员
自体CD34+造血干祖细胞是进行编辑的理想靶细胞,但其数量少并且难以分离,需要从患者外周血或者骨髓进行动员以获得足量造血干祖细胞。普通患者以粒细胞集落刺激因子(granulocyte colony- stimulating factor, G-CSF)作为动员剂,但是镰状红细胞贫血病人单独使用G-CSF会引起HbS的多聚化等一系列的反应,导致镰状红细胞危象甚至是多器官的衰竭[32]。小分子趋化因子受体CXCR4抑制剂普乐沙福(plerixafor)相比G-CSF是更安全的动员剂[33]。鉴于在临床上采集人造血干祖细胞难度较大,Yang等[34]、Song等[35]和Xie等[36]尝试将患者来源的成纤维细胞重编程为诱导多能干细胞(induced pluripotent stem cell, iPSC)后,利用CRISPER/Cas9对β-地中海贫血基因进行了纠正,获得了基因缺陷得以修复的iPSC细胞。虽然该方法存在一定的畸变风险,但仍是值得密切关注的研究方向。
2.2 编辑试剂的运载系统
采用何种传输系统将基因组编辑试剂送入靶细胞是建立临床转化平台中最核心的部分,也是最大的瓶颈之一。目前,传输系统分为非病毒型系统和病毒型载体,非病毒型系统有电穿孔,脂质体转染等方式,而病毒型载体有慢病毒载体、腺病毒载体以及腺相关病毒载体。其中慢病毒载体属于整合型病毒载体,而后两者属于非整合型病毒载体。相对于慢病毒等整合型载体,非整合型病毒载体或者电穿孔等方法作为瞬时导入策略,引起宿主基因组发生插入突变的风险较低,并可消除长期稳定表达编辑试剂所造成的宿主基因组不稳定性的风险,安全性更高[37],本文列举的多个研究组均采用这类方 法[17,18,20,38,39]。其中DeWitt等[18]和Dever等[20]都使用了Cas9蛋白与gRNA偶联在一起形成Cas9 RNP复合物的方法,以增加gRNA稳定性。最近的研究显示mRNA的电传孔可成功应用于108×以上的CD34+造血干祖细胞[40],以满足其中临床治疗中往往需要至少108×的CD34+造血干祖细胞的需求,说明电穿孔方法相比其他方法具有突出的优势。2.3 核酸酶的优化和脱靶效应的控制
相比ZFN、TALEN等核酸酶,Cas9核酸酶因分子量低更有优势。Cas9属于II型CRISPR-Cas系统,这一系统使用单一的Cas核酸酶和guide RNA即可发挥切割作用,体系简单易于转化。该领域进展迅速,新的家族成员不断被鉴定出来,如Cas12a,Cas12b,Cas13e,Cas13a和Cas13b等,其中Cas12a又称为Cpf1,因其分子量小,只需一段42~44 bp的核苷酸组成的单链RNA即可识别和切割DNA等优点而备受关注[41]。对sgRNA的化学修饰可以提高在原代造血细胞或者CD34+造血干祖细胞中的编辑效率[40]。此外其他系统如通过一对sgRNA引导的dCas9-FoK I系统,或对Cas9进行修饰等也可提高准确打靶的效率[9]。需要建立新的筛选方法,以求在骨髓移植前筛选出可能造成不良后果的脱靶编辑的造血干祖细胞。此外,还需借助CD34+人源化小鼠,人CD34+造血干祖细胞植入的NOD-SCID-γ小鼠等动物模型评估基因编辑后的干祖细胞的安全性,多潜能分化能力,自我更新能力以及诱发白血病的风险[42]。2.4 编辑后细胞的自基因骨髓移植
骨髓移植需要进行清髓处理以促进造血干祖细胞的植入,处理不当可能会导致病人死亡。需要对β-地中海贫血症病人的过量铁负荷的程度或者镰状红细胞贫血病人的器官损伤程度进行准确评估,继而选择个体化的清髓处理方法[42]。对β-地中海贫血症病人,可根据铁螯合剂应用史、门静脉纤维化以及肝脏MRI T2*等客观指标,对病人进行移植前危险度评分并选择合适的预处理方案。经典预处理方案是由白消安和环磷酰胺组成。I度和II度重型β-地中海贫血症患者预处理方案多采用白消安14 mg/kg,环磷酰胺200 mg/kg。为克服III度β-常在此经典预处理方案基础上加入塞替派、氟达拉滨、抗胸腺细胞免疫球蛋白或二羟马利兰等。镰状红细胞贫血病人的清髓方案也是根据病人的情况对以上标准清髓方案进行调整[32]。病人基因组编辑结合自基因骨髓移植因是自体细胞移植,无需使用多种免疫抑制剂或者低剂量放疗以抑制移植物抗宿主病,这也是这种方案相比传统的异基因骨髓移植的优势所在。目前骨髓移植价格昂贵,对医疗条件的要求很高,在发展中国家的推广面临大的挑战[32]。3 结语与展望
随着基因组编辑技术的飞速发展以及对β-血红蛋白病的发病机制的深入认识,对β-血红蛋白病的基因治疗重新成为人们的研究的热点。相比传统的以病毒载体表达正常血红蛋白的基因治疗策略,通过瞬时表达核酸酶造成DNA双链断裂缺口从而激活细胞内源性DNA损伤修复机制的基因组编辑策略表现出明显的优势(图1)。一方面,这种治疗策略能够避免传统基因治疗策略中病毒表达载体的导入和整合对病人基因组稳定性的不利影响;另一方面它还能通过瞬时转染引入核酸酶,实现对病人基因组的永久的改造。其中,利用HDR机制可修复β-血红蛋白患者自身的遗传缺陷,无论是使用ZFN核酸酶还是Cas9核酸酶均取得了成功。其中以Cas9 RNP复合物进行基因组编辑的方法因Cas9蛋白质转入细胞后,不需经过转录或者翻译过程而直接发挥作用,且同时可增加gRNA的稳定性而有一定优势。HDR效率很低的问题可被一些基因组编辑后的造血干细胞富集策略改善,但HDR策略需要针对每个病人的独特突变进行个性化编辑方案设计,临床转化的挑战相对较大。利用NHEJ机制可破坏对胎儿血红蛋白的表达具有关键抑制作用的分子,重新激活胎儿血红蛋白的表达继而缓解β-血红蛋白病人的症状。BCL11A、LRF、KLF1和MYB等可抑制胎儿血红蛋白表达的分子均为潜在的靶点,其中破坏BCL11A红系增强子是最有潜力的方向在于它只专一性的破坏BCL11A在红细胞中的表达,而对BCL11A在其他系统的功能没有影响,临床应用时副作用小。另通过基因组编辑技术模拟β珠蛋白基因座位有益的先天突变的方案也很有前景。NHEJ策略不需要针对每个病人的特定突变进行设计,作为针对不同病人的普适性方案,更易于向临床转化。综上所述,随着新的有效靶点的不断鉴定,新的编辑策略不断出现,基因组编辑方案向临床转化的可能性大大增加,它的下一步发展值得拭目以待,有望为病痛中的患者带来痊愈的福音。图1
新窗口打开|下载原图ZIP|生成PPT图1β-血红蛋白病人造血干祖细胞基因组编辑流程图
Fig. 1The flowchart of genome editing of HSPCs of β-hemoglobinopathies patients
对病人自体造血干祖细胞进行动员和采集后,在体外可根据病人的个体化特点分别选择HDR(homology-directed repair, HDR)或者NHEJ(nonhomologous end joining,NHEJ)策略进行基因组编辑,然后对编辑成功的造血干细胞进行选择或者富集,最后对病人进行自体造血干细胞移植。其中HDR策略利用CRISPR/Cas9、ZFN等核酸酶,结合供体DNA模板,利用细胞内的同源重组机制实现原位无痕修复,但是受限于同源重组的频率很低;NHEJ策略利用CRISPR/Cas9、ZFN等核酸酶造成DNA双链断裂缺口,利用细胞内的另一条修复机制即非同源末端连接机制破坏可调控β-globin表达或者功能发挥的一些关键分子或者产生一些有益突变,激活胎儿血红蛋白的表达,从而减缓β-血红蛋白病人的症状。
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URLPMID:28605432 [本文引用: 1]
Abstract Objectives: To describe the use of molecular diagnostic techniques for patients with hemoglobin disorders. Methods: A clinical scenario is presented in which molecular diagnosis is important for genetic counseling. Globin disorders, techniques for their diagnosis, and the role of molecular genetic testing in managing patients with these disorders are described in detail. Results: Hemoglobin disorders, including thalassemias and hemoglobinopathies, are among the commonest genetic diseases, and the clinical laboratory is essential for the diagnosis of patients with these abnormalities. Most disorders can be diagnosed with protein-based techniques such as electrophoresis and chromatography. Since severe syndromes can result due to inheritance of combinations of globin genetic disorders, genetic counseling is important to prevent adverse outcomes. Protein-based methods cannot always detect potentially serious thalassemia disorders; in particular, $\beta$-thalassemia may be masked in the presence of $\beta$-thalassemia. Deletional forms of $\beta$-thalassemia are also sometimes difficult to diagnose definitively with standard methods. Conclusions: Molecular genetic testing serves an important role in identifying individuals carrying thalassemia traits that can cause adverse outcomes in offspring. Furthermore, prenatal genetic testing can identify fetuses with severe globin phenotypes.
URLPMID:18991645 [本文引用: 1]
The genetic disorders of hemoglobin, the commonest monogenic diseases, occur at some of their highest frequencies in the developing countries, particularly those of Sub-Saharan Africa and Asia. Although progress towards their control and management continues to be made, the prospects for curing them, apart from marrow transplantation, remain uncertain. In many countries expertise and facilities for their control are extremely limited. Although a great deal can be done to help the situation by developing further North/South and South/South partnerships for disseminating better practice, the major problem for the future lies in the unwillingness of governments and international health agencies to accept that the hemoglobinopathies represent a health burden comparative to that of communicable and other major diseases. However, preliminary analyses suggest that, at least in the case of Asia, this may not be true. Further work of this type, together with more detailed frequency and economic data, is required to provide solid evidence for the health burden posed by the hemoglobin disorders, particularly in the developing world. Unless this is done, the increasingly large populations of patients with these diseases will continue to be neglected. <br/> <br/> <br/>
URLPMID:27909215 [本文引用: 1]
Allogeneic hematopoietic stem cell transplantation has been well established for several decades as gene replacement therapy for patients with thalassemia major, and now offers very high rates of cure for patients who have access to this therapy. Outcomes have improved tremendously over the last decade, even in high-risk patients. The limited data available suggests that the long-term outcome is also excellent, with a >90% survival rate, but for the best results, hematopoietic stem cell transplantation should be offered early, before any end organ damage occurs. However, access to this therapy is limited in more than half the patients by the lack of suitable donors. Inadequate hematopoietic stem cell transplantation services and the high cost of therapy are other reasons for this limited access, particularly in those parts of the world which have a high prevalence of this condition. As a result, fewer than 10% of eligible patients are actually able to avail of this therapy. Other options for curative therapies are therefore needed. Recently, gene correction of autologous hematopoietic stem cells has been successfully established using lentiviral vectors, and several clinical trials have been initiated. A gene editing approach to correct the $\beta$-globin mutation or disrupt the BCL11A gene to increase fetal hemoglobin production has also been reported, and is expected to be introduced in clinical trials soon. Curative possibilities for the major hemoglobin disorders are expanding. Providing access to these therapies around the world will remain a challenge.
URLPMID:27053533 [本文引用: 3]
Abstract Despite nearly complete understanding of the genetics of the 0205-hemoglobinopathies for several decades, definitive treatment options have lagged behind. Recent developments in technologies for facile manipulation of the genome (zinc finger nucleases, transcription activator-like effector nucleases, or clustered regularly interspaced short palindromic repeats-based nucleases) raise prospects for their clinical application. The use of genome-editing technologies in autologous CD34(+) hematopoietic stem and progenitor cells represents a promising therapeutic avenue for the 0205-globin disorders. Genetic correction strategies relying on the homology-directed repair pathway may repair genetic defects, whereas genetic disruption strategies relying on the nonhomologous end joining pathway may induce compensatory fetal hemoglobin expression. Harnessing the power of genome editing may usher in a second-generation form of gene therapy for the 0205-globin disorders. 0008 2016 by The American Society of Hematology.
URLPMID:26375765 [本文引用: 5]
The major disorders of β-globin, sickle cell disease and β-thalassemia, may be ameliorated by expression of the fetal gene paralog γ-globin. Uncertainty regarding the mechanisms repressing fetal hemoglobin in the adult stage has served as a puzzle of developmental gene regulation as well as a barrier to rational therapeutic design. Recent genome-wide association studies implicated the zinc-finger transcriptional repressor BCL11A in fetal hemoglobin regulation. Extensive genetic analyses have validated BCL11A as a potent repressor of fetal hemoglobin level. Studies of BCL11A exemplify how contextual gene regulation may often be the substrate for trait-associated common genetic variation. These discoveries have suggested novel rational approaches for the β-hemoglobin disorders including therapeutic genome editing.
URLPMID:27502996 [本文引用: 1]
Abstract The underlying basis of 0205-thalassemia pathology is the diminished 0205-globin synthesis leading to 02±-globin accumulation and premature apoptotic destruction of erythroblasts, causing oxidative stress-induced ineffective erythropoiesis, bone marrow hyperplasia, splenomegaly, and increased intestinal iron absorption with progressive iron overload. Better understanding of the molecular mechanisms underlying this disease led to the recognition of new targets with potential therapeutic utility. Agents such as JAK2 inhibitors and TGF-0205 ligand traps that reduce the ineffective erythropoiesis process are already being tested in clinical trials with promising results. Other agents that aim to reduce oxidative stress (activators of Foxo3, HRI-eIF2aP, Prx2, Hsp70, and PK anti-oxidant systems and inhibitors of HO-1) and to decrease iron overload (hepcidin agonists, erythroferrone inhibitors and exogenous transferrin) are also under experimental investigation. Significant progress has also been made in the area of allogeneic hematopoietic stem cell transplantation with several ongoing clinical trials examining new condition regimens as well as different donor selection and stem cell source options. Gene therapy has reached a critical point and phase 1 clinical trials have recently been launched to examine the effectiveness and especially long term safety. Epigenetic manipulation and genomic editing of the 0206- or 0205-globin gene are novel and promising experimental gene therapy approaches for 0205-thalassemia giving hope for cure for this chronic disease. This review outlines the key points of the molecular mechanisms underlying 0205-thalassemia in relation to the development of new therapies and an update is given both at the pre-clinical and clinical level. Am. J. Hematol. 91:1135-1145, 2016. 0008
URLPMID:28377044 [本文引用: 1]
Abstract β-Thalassemia and sickle cell disease (SCD) are the world's two most widely disseminated hereditary hemoglobinopathies. β-Thalassemia originated in the Mediterranean, Middle Eastern, and Asian regions, and SCD originated in central Africa. However, subsequent population migration means that these two diseases are now global and thus constitute a growing health problem in many countries. Despite remarkable improvements in medical care for patients with β-hemoglobinopathies, there is still only one definitive treatment option: allogeneic hematopoietic stem cell (HSC) transplantation. The development of gene therapy for β-hemoglobinopathies has been justified by (1) the limited availability of human leukocyte antigen (HLA)-identical donors, (2) the narrow window of application of HSC transplantation to the youngest patients, and (3) recent advances in HSC-based gene therapy. The huge ongoing efforts in translational medicine and the high number of related publications show that gene therapy has the potential to become the treatment of choice for patients who lack either an HLA genoidentical sibling or an alternative, medically acceptable donor. In this dynamic scientific context, we first summarize the main steps toward clinical translation of02this therapeutic approach and then discuss novel lentiviral- and genome editing-based treatment strategies for β-hemoglobinopathies. Copyright 08 2017 The American Society of Gene and Cell Therapy. Published by Elsevier Inc. All rights reserved.
URLPMID:24584096 [本文引用: 1]
Abstract Targeted genome editing using engineered nucleases has rapidly gone from being a niche technology to a mainstream method used by many biological researchers. This widespread adoption has been largely fueled by the emergence of the clustered, regularly interspaced, short palindromic repeat (CRISPR) technology, an important new approach for generating RNA-guided nucleases, such as Cas9, with customizable specificities. Genome editing mediated by these nucleases has been used to rapidly, easily and efficiently modify endogenous genes in a wide variety of biomedically important cell types and in organisms that have traditionally been challenging to manipulate genetically. Furthermore, a modified version of the CRISPR-Cas9 system has been developed to recruit heterologous domains that can regulate endogenous gene expression or label specific genomic loci in living cells. Although the genome-wide specificities of CRISPR-Cas9 systems remain to be fully defined, the power of these systems to perform targeted, highly efficient alterations of genome sequence and gene expression will undoubtedly transform biological research and spur the development of novel molecular therapeutics for human disease.
URLPMID:27866654 [本文引用: 3]
The CRISPR-Cas9 RNA-guided DNA endonuclease has contributed to an explosion of advances in the life sciences that have grown from the ability to edit genomes within living cells. In this Review, we summarize CRISPR-based technologies that enable mammalian genome editing and their various applications. We describe recent developments that extend the generality, DNA specificity, product selectivity, and fundamental capabilities of natural CRISPR systems, and we highlight some of the remarkable advancements in basic research, biotechnology, and therapeutics science that these developments have facilitated.
URL [本文引用: 1]
CRISPR/Cas 系统是广泛存在于细菌和古生菌中的适应性免疫系统，用来抵抗外来病毒或质粒的入侵。近几年，由Ⅱ型CRISPR/Cas适应性免疫系统改造而来的 CRISPR/Cas9基因组编辑技术蓬勃发展，被广泛地应用于生命科学研究的各个领域，并取得了革命性的变化。文章主要综述了 CRISPR/Cas9基因组编辑技术的起源与发展及在生命科学各研究领域的应用，重点介绍了该系统在人类疾病基因治疗方面的最新应用及脱靶效应，以期为 相关领域的科研人员提供参考。
URL [本文引用: 1]
CRISPR/Cas 系统是广泛存在于细菌和古生菌中的适应性免疫系统，用来抵抗外来病毒或质粒的入侵。近几年，由Ⅱ型CRISPR/Cas适应性免疫系统改造而来的 CRISPR/Cas9基因组编辑技术蓬勃发展，被广泛地应用于生命科学研究的各个领域，并取得了革命性的变化。文章主要综述了 CRISPR/Cas9基因组编辑技术的起源与发展及在生命科学各研究领域的应用，重点介绍了该系统在人类疾病基因治疗方面的最新应用及脱靶效应，以期为 相关领域的科研人员提供参考。
URLPMID:26212079 [本文引用: 1]
Abstract Hemophilia A is an X-linked genetic disorder caused by mutations in the F8 gene, which encodes the blood coagulation factor VIII. Almost half of all severe hemophilia A cases result from two gross (140-kbp or 600-kbp) chromosomal inversions that involve introns 1 and 22 of the F8 gene, respectively. We derived induced pluripotent stem cells (iPSCs) from patients with these inversion genotypes and used CRISPR-Cas9 nucleases to revert these chromosomal segments back to the WT situation. We isolated inversion-corrected iPSCs with frequencies of up to 6.7% without detectable off-target mutations based on whole-genome sequencing or targeted deep sequencing. Endothelial cells differentiated from corrected iPSCs expressed the F8 gene and functionally rescued factor VIII deficiency in an otherwise lethal mouse model of hemophilia. Our results therefore provide a proof of principle for functional correction of large chromosomal rearrangements in patient-derived iPSCs and suggest potential therapeutic applications.
URLPMID:24597865 [本文引用: 1]
Abstract BACKGROUND: CCR5 is the major coreceptor for human immunodeficiency virus (HIV). We investigated whether site-specific modification of the gene ("gene editing")--in this case, the infusion of autologous CD4 T cells in which the CCR5 gene was rendered permanently dysfunctional by a zinc-finger nuclease (ZFN)--is safe. METHODS: We enrolled 12 patients in an open-label, nonrandomized, uncontrolled study of a single dose of ZFN-modified autologous CD4 T cells. The patients had chronic aviremic HIV infection while they were receiving highly active antiretroviral therapy. Six of them underwent an interruption in antiretroviral treatment 4 weeks after the infusion of 10 billion autologous CD4 T cells, 11 to 28% of which were genetically modified with the ZFN. The primary outcome was safety as assessed by treatment-related adverse events. Secondary outcomes included measures of immune reconstitution and HIV resistance. RESULTS: One serious adverse event was associated with infusion of the ZFN-modified autologous CD4 T cells and was attributed to a transfusion reaction. The median CD4 T-cell count was 1517 per cubic millimeter at week 1, a significant increase from the preinfusion count of 448 per cubic millimeter (P<0.001). The median concentration of CCR5-modified CD4 T cells at 1 week was 250 cells per cubic millimeter. This constituted 8.8% of circulating peripheral-blood mononuclear cells and 13.9% of circulating CD4 T cells. Modified cells had an estimated mean half-life of 48 weeks. During treatment interruption and the resultant viremia, the decline in circulating CCR5-modified cells (-1.81 cells per day) was significantly less than the decline in unmodified cells (-7.25 cells per day) (P=0.02). HIV RNA became undetectable in one of four patients who could be evaluated. The blood level of HIV DNA decreased in most patients. CONCLUSIONS: CCR5-modified autologous CD4 T-cell infusions are safe within the limits of this study. (Funded by the National Institute of Allergy and Infectious Diseases and others; ClinicalTrials.gov number, NCT00842634 .).
URL [本文引用: 1]
基因治疗是将外源正常基因通过一定方式导入人体靶细胞以纠正或补偿因基因缺陷和异常引起的疾病,从而达到治疗目的.因此,基因治疗的技术方法在研究持续感染HIV-1或潜伏感染HIV-l原病毒患者的治疗中具有重大的现实意义.目前,现有的基因治疗方法存在识别靶向位点有限及脱靶几率大等主要问题.最新研究表明来源于细菌和古菌的规律间隔成簇短回文重复序列及其相关核酸酶9系统[Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated nuclease 9(Cas9),CRISPR/Cas9]已被成功改造成基因组定点编辑工具.因此,如何利用CRISPR/Cas9系统实现对HIV-1病毒基因组进行高效靶向修饰,从而达到治疗HIV-1感染病患的目的已经成为当前研究的热点.本文参考最新国内外研究成果,重点介绍了CRISPR/Cas9基因组编辑技术在HIV-1感染疾病治疗中的应用,主要包括CCR5基因编辑、清除HIV-1原病毒以及活化HIV-1原病毒,以期为HIV-1感染疾病的预防与治疗提供重要研究参考.
URL [本文引用: 1]
基因治疗是将外源正常基因通过一定方式导入人体靶细胞以纠正或补偿因基因缺陷和异常引起的疾病,从而达到治疗目的.因此,基因治疗的技术方法在研究持续感染HIV-1或潜伏感染HIV-l原病毒患者的治疗中具有重大的现实意义.目前,现有的基因治疗方法存在识别靶向位点有限及脱靶几率大等主要问题.最新研究表明来源于细菌和古菌的规律间隔成簇短回文重复序列及其相关核酸酶9系统[Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated nuclease 9(Cas9),CRISPR/Cas9]已被成功改造成基因组定点编辑工具.因此,如何利用CRISPR/Cas9系统实现对HIV-1病毒基因组进行高效靶向修饰,从而达到治疗HIV-1感染病患的目的已经成为当前研究的热点.本文参考最新国内外研究成果,重点介绍了CRISPR/Cas9基因组编辑技术在HIV-1感染疾病治疗中的应用,主要包括CCR5基因编辑、清除HIV-1原病毒以及活化HIV-1原病毒,以期为HIV-1感染疾病的预防与治疗提供重要研究参考.
URL [本文引用: 1]
基因组编辑技术的飞速发展,尤其是近年来CRISPR/Cas9基因组编辑体系的出现,使得研究人员能高效地在细胞系和动物模型中对基因组进行精确编辑。基于基因组编辑技术的各种实验研究平台被相继开发,包括通过在细胞系中引入疾病相关突变位点建立疾病模型,通过高通量筛选寻找导致肿瘤耐药性的突变基因,通过体内原位靶向致病基因并修改突变进行基因治疗等。这些基因组编辑技术研究平台极大推动了精准医学研究领域的发展。本文对基因组编辑技术在精准医学领域的基础研究、转化应用、目前存在的问题以及未来发展的方向进行了讨论。
URL [本文引用: 1]
基因组编辑技术的飞速发展,尤其是近年来CRISPR/Cas9基因组编辑体系的出现,使得研究人员能高效地在细胞系和动物模型中对基因组进行精确编辑。基于基因组编辑技术的各种实验研究平台被相继开发,包括通过在细胞系中引入疾病相关突变位点建立疾病模型,通过高通量筛选寻找导致肿瘤耐药性的突变基因,通过体内原位靶向致病基因并修改突变进行基因治疗等。这些基因组编辑技术研究平台极大推动了精准医学研究领域的发展。本文对基因组编辑技术在精准医学领域的基础研究、转化应用、目前存在的问题以及未来发展的方向进行了讨论。
URL [本文引用: 1]
利用功能缺失型(Loss-of-function)或者功能获得型(Gain-of-function) 策略高通量筛选功能基因,是研究人员快速寻找调控特定表型的重要或关键基因的主要方法。RNA干扰(RNA interference,RNAi)的遗传筛选方法因操作简单、成本相对较低等优势,尽管已经得到了广泛的应用,然而其抑制效果不完全、脱靶效应明显等劣势依然存在。近年来兴起的CRISPR/Cas9 (Clustered regularly interspaced short palindromic repeat sequences/ CRISPR-associated protein 9)技术能快速、简便、准确地实现基因组敲除等编辑功能,因而成为一种强大的遗传筛选工具;在各种细胞系、人和小鼠及斑马鱼等多种模式动物中,大规模运用该方法筛选功能基因已经取得了巨大成功。本文总结了CRISPR/Cas9技术的特点,将其与传统基因工程方法进行了分析比较,回顾了近期相关的高通量功能基因筛选工作,最后探讨了该技术未来的发展趋势。
URL [本文引用: 1]
利用功能缺失型(Loss-of-function)或者功能获得型(Gain-of-function) 策略高通量筛选功能基因,是研究人员快速寻找调控特定表型的重要或关键基因的主要方法。RNA干扰(RNA interference,RNAi)的遗传筛选方法因操作简单、成本相对较低等优势,尽管已经得到了广泛的应用,然而其抑制效果不完全、脱靶效应明显等劣势依然存在。近年来兴起的CRISPR/Cas9 (Clustered regularly interspaced short palindromic repeat sequences/ CRISPR-associated protein 9)技术能快速、简便、准确地实现基因组敲除等编辑功能,因而成为一种强大的遗传筛选工具;在各种细胞系、人和小鼠及斑马鱼等多种模式动物中,大规模运用该方法筛选功能基因已经取得了巨大成功。本文总结了CRISPR/Cas9技术的特点,将其与传统基因工程方法进行了分析比较,回顾了近期相关的高通量功能基因筛选工作,最后探讨了该技术未来的发展趋势。
URLPMID:27053532 [本文引用: 1]
Gene editing enables the site-specific modification of the genome. These technologies have rapidly advanced such that they have entered common use in experimental hematology to investigate genetic function. In addition, genome editing is becoming increasingly plausible as a treatment modality to rectify genetic blood disorders and improve cellular therapies. Genome modification typically ensues from site-specific double-strand breaks and may result in a myriad of outcomes. Even single-strand nicks and targeted biochemical modifications that do not permanently alter the DNA sequence (epigenome editing) may be powerful instruments. In this review, we examine the various technologies, describe their advantages and shortcomings for engendering useful genetic alterations, and consider future prospects for genome editing to impact hematology.
URLPMID:25733580 [本文引用: 3]
Sickle cell disease (SCD) is characterized by a single point mutation in the seventh codon of the $\beta$-globin gene. Site-specific correction of the sickle mutation in hematopoietic stem cells would allow for permanent production of normal red blood cells. Using zinc-finger nucleases (ZFNs) designed to flank the sickle mutation, we demonstrate efficient targeted cleavage at the $\beta$-globin locus with minimal off-target modification. By co-delivering a homologous donor template (either an integrase-defective lentiviral vector or a DNA oligonucleotide), high levels of gene modification were achieved in CD34(+) hematopoietic stem and progenitor cells. Modified cells maintained their ability to engraft NOD/SCID/IL2ry(null) mice and to produce cells from multiple lineages, although with a reduction in the modification levels relative to the in vitro samples. Importantly, ZFN-driven gene correction in CD34(+) cells from the bone marrow of patients with SCD resulted in the production of wild-type hemoglobin tetramers.
URLPMID:27733558 [本文引用: 3]
Genetic diseases of blood cells are prime candidates for treatment through ex vivo gene editing of CD34+ hematopoietic stem/progenitor cells (HSPCs), and a variety of technologies have been proposed to treat these disorders. Sickle cell disease (SCD) is a recessive genetic disorder caused by a single-nucleotide polymorphism in the $\beta$-globin gene (HBB). Sickle hemoglobin damages erythrocytes, causing vasoocclusion, severe pain, progressive organ damage, and premature death. We optimize design and delivery parameters of a ribonucleoprotein (RNP) complex comprising Cas9 protein and unmodified single guide RNA, together with a single-stranded DNA oligonucleotide donor (ssODN), to enable efficient replacement of the SCD mutation in human HSPCs. Corrected HSPCs from SCD patients produced less sickle hemoglobin RNA and protein and correspondingly increased wild-type hemoglobin when differentiated into erythroblasts. When engrafted into immunocompromised mice, ex vivo treated human HSPCs maintain SCD gene edits throughout 16 weeks at a level likely to have clinical benefit. These results demonstrate that an accessible approach combining Cas9 RNP with an ssODN can mediate efficient HSPC genome editing, enables investigator-led exploration of gene editing reagents in primary hematopoietic stem cells, and suggests a path toward the development of new gene editing treatments for SCD and other hematopoietic diseases.
URLPMID:27782131 [本文引用: 3]
Abstract The blood disorder, 0205-thalassaemia, is considered an attractive target for gene correction. Site-specific triplex formation has been shown to induce DNA repair and thereby catalyse genome editing. Here we report that triplex-forming peptide nucleic acids (PNAs) substituted at the 0206 position plus stimulation of the stem cell factor (SCF)/c-Kit pathway yielded high levels of gene editing in haematopoietic stem cells (HSCs) in a mouse model of human 0205-thalassaemia. Injection of thalassemic mice with SCF plus nanoparticles containing 0206PNAs and donor DNAs ameliorated the disease phenotype, with sustained elevation of blood haemoglobin levels into the normal range, reduced reticulocytosis, reversal of splenomegaly and up to 7% 0205-globin gene correction in HSCs, with extremely low off-target effects. The combination of nanoparticle delivery, next generation 0206PNAs and SCF treatment may offer a minimally invasive treatment for genetic disorders of the blood that can be achieved safely and simply by intravenous administration.
URLPMID:27820943 [本文引用: 3]
Abstract The 0205-haemoglobinopathies, such as sickle cell disease and 0205-thalassaemia, are caused by mutations in the 0205-globin (HBB) gene and affect millions of people worldwide. Ex vivo gene correction in patient-derived haematopoietic stem cells followed by autologous transplantation could be used to cure 0205-haemoglobinopathies. Here we present a CRISPR/Cas9 gene-editing system that combines Cas9 ribonucleoproteins and adeno-associated viral vector delivery of a homologous donor to achieve homologous recombination at the HBB gene in haematopoietic stem cells. Notably, we devise an enrichment model to purify a population of haematopoietic stem and progenitor cells with more than 90% targeted integration. We also show efficient correction of the Glu6Val mutation responsible for sickle cell disease by using patient-derived stem and progenitor cells that, after differentiation into erythrocytes, express adult 0205-globin (HbA) messenger RNA, which confirms intact transcriptional regulation of edited HBB alleles. Collectively, these preclinical studies outline a CRISPR-based methodology for targeting haematopoietic stem cells by homologous recombination at the HBB locus to advance the development of next-generation therapies for 0205-haemoglobinopathies.
URLPMID:25971621 [本文引用: 1]
Genetic disorders resulting from defects in the adult globin genes are among the most common inherited diseases. Symptoms worsen from birth as fetal γ-globin expression is silenced. Genome editing could permit the introduction of beneficial single-nucleotide variants to ameliorate symptoms. Here, as proof of concept, we introduce the naturally occurring Hereditary Persistance of Fetal Haemoglobin (HPFH) -175T>C point mutation associated with elevated fetal γ-globin into erythroid cell lines. We show that this mutation increases fetal globin expression through de novo recruitment of the activator TAL1 to promote chromatin looping of distal enhancers to the modified γ-globin promoter.
URLPMID:27525524 [本文引用: 1]
Abstract Disorders resulting from mutations in the hemoglobin subunit beta gene (HBB; which encodes 0205-globin), mainly sickle cell disease (SCD) and 0205-thalassemia, become symptomatic postnatally as fetal 0206-globin expression from two paralogous genes, hemoglobin subunit gamma 1 (HBG1) and HBG2, decreases and adult 0205-globin expression increases, thereby shifting red blood cell (RBC) hemoglobin from the fetal (referred to as HbF or 02±202062) to adult (referred to as HbA or 02±202052) form. These disorders are alleviated when postnatal expression of fetal 0206-globin is maintained. For example, in hereditary persistence of fetal hemoglobin (HPFH), a benign genetic condition, mutations attenuate 0206-globin-to-0205-globin switching, causing high-level HbF expression throughout life. Co-inheritance of HPFH with 0205-thalassemia- or SCD-associated gene mutations alleviates their clinical manifestations. Here we performed CRISPR-Cas9-mediated genome editing of human blood progenitors to mutate a 13-nt sequence that is present in the promoters of the HBG1 and HBG2 genes, thereby recapitulating a naturally occurring HPFH-associated mutation. Edited progenitors produced RBCs with increased HbF levels that were sufficient to inhibit the pathological hypoxia-induced RBC morphology found in SCD. Our findings identify a potential DNA target for genome-editing-mediated therapy of 0205-hemoglobinopathies.
URLPMID:28190778 [本文引用: 1]
Abstract Gene therapy may finally lead to a cure for sickle cell disease, the most common form of inherited blood disorders. In particular, genome editing aimed at increasing fetal hemoglobin production or replacing the sickle cell mutation with a wild-type sequence has gained momentum. Recent publications demonstrate feasibility in human hematopoietic stem and progenitor cells (HSPCs) when subsequently evaluated in mouse transplantation studies. CRISPR/Cas9 or zinc finger nucleases (ZFNs) have proven to be useful tools to delete or replace sequences involved in the production of hemoglobin.
URLPMID:4644101 [本文引用: 1]
Abstract Enhancers, critical determinants of cellular identity, are commonly recognized by correlative chromatin marks and gain-of-function potential, although only loss-of-function studies can demonstrate their requirement in the native genomic context. Previously, we identified an erythroid enhancer of human BCL11A, subject to common genetic variation associated with the fetal haemoglobin level, the mouse orthologue of which is necessary for erythroid BCL11A expression. Here we develop pooled clustered regularly interspaced palindromic repeat (CRISPR)-Cas9 guide RNA libraries to perform in situ saturating mutagenesis of the human and mouse enhancers. This approach reveals critical minimal features and discrete vulnerabilities of these enhancers. Despite conserved function of the composite enhancers, their architecture diverges. The crucial human sequences appear to be primate-specific. Through editing of primary human progenitors and mouse transgenesis, we validate the BCL11A erythroid enhancer as a target for fetal haemoglobin reinduction. The detailed enhancer map will inform therapeutic genome editing, and the screening approach described here is generally applicable to functional interrogation of non-coding genomic elements.
URLPMID:5363298 [本文引用: 1]
To develop an effective and sustainable cell therapy for sickle cell disease (SCD), we investigated the feasibility of targeted disruption of theBCL11Agene, either within exon 2 or at the GATAA motif in the intronic erythroid-specific enhancer, using zinc finger nucleases in human bone marrow (BM) CD34+hematopoietic stem and progenitor cells (HSPCs). Both targeting strategies upregulated fetal globin expression in erythroid cells to levels predicted to inhibit hemoglobin S polymerization. However, complete inactivation ofBCL11Aresulting from bi-allelic frameshift mutations inBCL11Aexon 2 adversely affected erythroid enucleation. In contrast, bi-allelic disruption of the GATAA motif in the erythroid enhancer ofBCL11Adid not negatively impact enucleation. Furthermore,BCL11Aexon 2-edited BM-CD34+cells demonstrated a significantly reduced engraftment potential in immunodeficient mice. Such an adverse effect on HSPC function was not observed uponBCL11Aerythroid-enhancer GATAA motif editing, because enhancer-edited CD34+cells achieved robust long-term engraftment and gave rise to erythroid cells with elevated levels of fetal globin expression when chimeric BM was cultured ex vivo. Altogether, our results support further clinical development of theBCL11Aerythroid-specific enhancer editing in BM-CD34+HSPCs as an autologous stem cell therapy in SCD patients.
URLPMID:26080908 [本文引用: 1]
RNA interference (RNAi) technology using short hairpin RNAs (shRNAs) expressed via RNA polymerase (pol) III promoters has been widely exploited to modulate gene expression in a variety of mammalian cell types. For certain applications, such as lineage-specific knockdown, embedding targeting sequences into pol II-driven microRNA (miRNA) architecture is required. Here, using the potential therapeutic target BCL11A, we demonstrate that pol III-driven shRNAs lead to significantly increased knockdown but also increased cytotoxcity in comparison to pol II-driven miRNA adapted shRNAs (shRNA miR ) in multiple hematopoietic cell lines. We show that the two expression systems yield mature guide strand sequences that differ by a 465bp shift. This results in alternate seed sequences and consequently influences the efficacy of target gene knockdown. Incorporating a corresponding 465bp shift into the guide strand of shRNA miR s resulted in improved knockdown efficiency of BCL11A. This was associated with a significant de-repression of the hemoglobin target of BCL11A, human γ-globin or the murine homolog Hbb-y. Our results suggest the requirement for optimization of shRNA sequences upon incorporation into a miRNA backbone. These findings have important implications in future design of shRNA miR s for RNAi-based therapy in hemoglobinopathies and other diseases requiring lineage-specific expression of gene silencing sequences.
URLPMID:26816381 [本文引用: 2]
Abstract Genes encoding human 0205-type globin undergo a developmental switch from embryonic to fetal to adult-type expression. Mutations in the adult form cause inherited hemoglobinopathies or globin disorders, including sickle cell disease and thalassemia. Some experimental results have suggested that these diseases could be treated by induction of fetal-type hemoglobin (HbF). However, the mechanisms that repress HbF in adults remain unclear. We found that the LRF/ZBTB7A transcription factor occupies fetal 0206-globin genes and maintains the nucleosome density necessary for 0206-globin gene silencing in adults, and that LRF confers its repressive activity through a NuRD repressor complex independent of the fetal globin repressor BCL11A. Our study may provide additional opportunities for therapeutic targeting in the treatment of hemoglobinopathies. Copyright 0008 2016, American Association for the Advancement of Science.
URLPMID:26320100 [本文引用: 1]
Abstract Fetal hemoglobin (HbF, 02±202062) induction is a well-validated strategy for sickle cell disease (SCD) treatment. Using a small-molecule screen, we found that UNC0638, a selective inhibitor of EHMT1 and EHMT2 histone methyltransferases, induces 0206-globin expression. EHMT1/2 catalyze mono- and dimethylation of lysine 9 on histone 3 (H3K9), raising the possibility that H3K9Me2, a repressive chromatin mark, plays a role in silencing 0206-globin expression. In primary human adult erythroid cells, UNC0638 and EHMT1 or EHMT2 short hairpin RNA-mediated knockdown significantly increased 0206-globin expression, HbF synthesis, and the percentage of cells expressing HbF. At effective concentrations, UNC0638 did not alter cell morphology, proliferation, or erythroid differentiation of primary human CD34(+) hematopoietic stem and progenitor cells in culture ex vivo. In murine erythroleukemia cells, UNC0638 and Ehmt2 CRISPR/Cas9-mediated knockout both led to a marked increase in expression of embryonic 0205-globin genes Hbb-0208y and Hbb-0205h1. In primary human adult erythroblasts, chromatin immunoprecipitation followed by sequencing analysis revealed that UNC0638 treatment leads to genome-wide depletion in H3K9Me2 and a concomitant increase in the activating mark H3K9Ac, which was especially pronounced at the 0206-globin gene region. In RNA-sequencing analysis of erythroblasts, 0206-globin genes were among the most significantly upregulated genes by UNC0638. Further increase in 0206-globin expression in primary human adult erythroid cells was achieved by combining EHMT1/2 inhibition with the histone deacetylase inhibitor entinostat or hypomethylating agent decitabine. Our data provide genetic and pharmacologic evidence that EHMT1 and EHMT2 are epigenetic regulators involved in 0206-globin repression and represent a novel therapeutic target for SCD. 0008 2015 by The American Society of Hematology.
URLPMID:23798711 [本文引用: 1]
Abstract Reactivation of fetal hemoglobin (HbF) holds therapeutic potential for sickle cell disease and $\beta$-thalassemias. In human erythroid cells and hematopoietic organs, LIN28B and its targeted let-7 microRNA family, demonstrate regulated expression during the fetal-to-adult developmental transition. To explore the effects of LIN28B in human erythroid cell development, lentiviral transduction was used to knockdown LIN28B expression in erythroblasts cultured from human umbilical cord CD34+ cells. The subsequent reduction in LIN28B expression caused increased expression of let-7 and significantly reduced HbF expression. Conversely, LIN28B overexpression in cultured adult erythroblasts reduced the expression of let-7 and significantly increased HbF expression. Cellular maturation was maintained including enucleation. LIN28B expression in adult erythroblasts increased the expression of y-globin, and the HbF content of the cells rose to levels >30% of their hemoglobin. Expression of carbonic anhydrase I, glucosaminyl (N-acetyl) transferase 2, and miR-96 (three additional genes marking the transition from fetal-to-adult erythropoiesis) were reduced by LIN28B expression. The transcription factor BCL11A, a well-characterized repressor of y-globin expression, was significantly down-regulated. Independent of LIN28B, experimental suppression of let-7 also reduced BCL11A expression and significantly increased HbF expression. LIN28B expression regulates HbF levels and causes adult human erythroblasts to differentiate with a more fetal-like phenotype.
URLPMID:29159826 [本文引用: 1]
Abstract 0205-hemoglobin disorders, such as 0205-thalassemia and sickle cell anemia are among the most prevalent inherited genetic disorders worldwide. These disorders are caused by mutations in the gene encoding hemoglobin-0205 (HBB), a vital protein found in red blood cells (RBCs) that carries oxygen from lungs to all parts of the human body. As a consequence, there has been an enduring interest in this field in formulating therapeutic strategies for the treatment of these diseases. Currently, there is no cure available for hemoglobin disorders, although, some patients have been treated with bone marrow transplantation, whose scope is limited because of the difficulty in finding a histocompatible donor and also due to transplant-associated clinical complications that can arise during the treatment. On account of these constraints, reactivation of fetal hemoglobin (HbF) synthesis holds immense promise and is a viable strategy to alleviate the symptoms of 0205-hemoglobin disorders. Development of new genomic tools has led to the identification of important natural genetic modifiers of hemoglobin switching which include BCL11A, KLF1, HBSIL-MYB, LRF, LSD1, LDB1, histone deacetylases 1 and 2 (HDAC1 and HDAC2). miRNAs are also promising therapeutic targets for development of more effective strategies for the induction of HbF production. Many new small molecule pharmacological inducers of HbF production are already under pre-clinical and clinical development. Furthermore, recent advancements in gene and cell therapy includes targeted genome editing and iPS cell technologies, both of which utilizes a patient's own cells, are emerging as extremely promising approaches for significantly reducing the burden of 0205-hemoglobin disorders. This article is protected by copyright. All rights reserved.
URLPMID:27264182 [本文引用: 1]
Abstract Erythroid cell commitment and differentiation proceed through activation of a lineage-restricted transcriptional network orchestrated by a group of well characterized genes. However, the minimal set of factors necessary for instructing red blood cell (RBC) development remains undefined. We employed a screen for transcription factors allowing direct lineage reprograming from fibroblasts to induced erythroid progenitors/precursors (iEPs). We show that Gata1, Tal1, Lmo2, and c-Myc (GTLM) can rapidly convert murine and human fibroblasts directly to iEPs. The transcriptional signature of murine iEPs resembled mainly that of primitive erythroid progenitors in the yolk sac, whereas addition of Klf1 or Myb to the GTLM cocktail resulted in iEPs with a more adult-type globin expression pattern. Our results demonstrate that direct lineage conversion is a suitable platform for defining and studying the core factors inducing the different waves of erythroid development.
URLPMID:22553502 [本文引用: 3]
The globally widespread single-gene disorders $\beta$-thalassemia and sickle cell anemia (SCA) can only be cured by allogeneic hematopoietic stem cell transplantation (HSCT). HSCT treatment of thalassemia has substantially improved over the last two decades, with advancements in preventive strategies, control of transplant-related complications, and preparative regimens. A risk class-based transplantation approach results in disease-free survival probabilities of 90%, 84%, and 78% for class 1, 2, and 3 thalassemia patients, respectively. Because of disease advancement, adult thalassemia patients have a higher risk for transplant-related toxicity and a 65% cure rate. Patients without matched donors could benefit from haploidentical mother-to-child transplantation. There is a high cure rate for children with SCA who receive HSCT following myeloablative conditioning protocols. Novel non-myeloablative transplantation protocols could make HSCT available to adult SCA patients who were previously excluded from allogeneic stem cell transplantation.
URLPMID:26089395 [本文引用: 1]
Globin gene therapy requires abundant numbers of highly engraftable, autologous hematopoietic stem cells expressing curative levels of β-globin on differentiation. In this study, CD34+ cells from 31 thalassemic patients mobilized with hydroxyurea+granulocyte colony-stimulating factor (G-CSF), G-CSF, Plerixafor, or Plerixafor+G-CSF were transduced with the TNS9.3.55 β-globin lentivector and compared for transducibility and globin expression in vitro, as well as engraftment potential in a xenogeneic model after partial myeloablation. Transduction efficiency and vector copy number (VCN) averaged 48.4 ± 2.8% and 1.91 ± 0.04, respectively, whereas expression approximated the one-copy normal β-globin output. Plerixafor+G-CSF cells produced the highest β-globin expression/VCN. Long-term multilineage engraftment and persistent VCN and vector expression was encountered in all xenografted groups, with Plerixafor+G-CSF-mobilized cells achieving superior short-term engraftment rates, with similar numbers of CD34+ cells transplanted. Overall, Plerixafor+G-CSF not only allows high CD34+ cell yields but also provides increased β-globin expression/VCN and enhanced early human chimerism under nonmyeloablative conditions, thus representing an optimal graft for thalassemia gene therapy.
URLPMID:4729555 [本文引用: 1]
Abstract Conventional primed human embryonic stem cells and induced pluripotent stem cells (iPSCs) exhibit molecular and biological characteristics distinct from pluripotent stem cells in the na0104ve state. Although na0104ve pluripotent stem cells show much higher levels of self-renewal ability and multidifferentiation capacity, it is unknown whether na0104ve iPSCs can be generated directly from patient somatic cells and will be superior to primed iPSCs. In the present study, we used an established 5i/L/FA system to directly reprogram fibroblasts of a patient with 0205-thalassemia into transgene-free na0104ve iPSCs with molecular signatures of ground-state pluripotency. Furthermore, these na0104ve iPSCs can efficiently produce cross-species chimeras. Importantly, using the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 nuclease genome editing system, these na0104ve iPSCs exhibit significantly improved gene-correction efficiencies compared with the corresponding primed iPSCs. Furthermore, human na0104ve iPSCs could be directly generated from noninvasively collected urinary cells, which are easily acquired and thus represent an excellent cell resource for further clinical trials. Therefore, our findings demonstrate the feasibility and superiority of using patient-specific iPSCs in the na0104ve state for disease modeling, gene editing, and future clinical therapy. SIGNIFICANCE: In the present study, transgene-free na0104ve induced pluripotent stem cells (iPSCs) directly converted from the fibroblasts of a patient with 0205-thalassemia in a defined culture system were generated. These na0104ve iPSCs, which show ground-state pluripotency, exhibited significantly improved single-cell cloning ability, recovery capacity, and gene-targeting efficiency compared with conventional primed iPSCs. These results provide an improved strategy for personalized treatment of genetic diseases such as 0205-thalassemia. 0008AlphaMed Press.
URLPMID:25517294 [本文引用: 1]
Abstract The generation of beta-thalassemia (0205-Thal) patient-specific induced pluripotent stem cells (iPSCs), subsequent homologous recombination-based gene correction of disease-causing mutations/deletions in the 0205-globin gene (HBB), and their derived hematopoietic stem cell (HSC) transplantation offers an ideal therapeutic solution for treating this disease. However, the hematopoietic differentiation efficiency of gene-corrected 0205-Thal iPSCs has not been well evaluated in the previous studies. In this study, we used the latest gene-editing tool, clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated 9 (Cas9), to correct 0205-Thal iPSCs; gene-corrected cells exhibit normal karyotypes and full pluripotency as human embryonic stem cells (hESCs) showed no off-targeting effects. Then, we evaluated the differentiation efficiency of the gene-corrected 0205-Thal iPSCs. We found that during hematopoietic differentiation, gene-corrected 0205-Thal iPSCs showed an increased embryoid body ratio and various hematopoietic progenitor cell percentages. More importantly, the gene-corrected 0205-Thal iPSC lines restored HBB expression and reduced reactive oxygen species production compared with the uncorrected group. Our study suggested that hematopoietic differentiation efficiency of 0205-Thal iPSCs was greatly improved once corrected by the CRISPR/Cas9 system, and the information gained from our study would greatly promote the clinical application of 0205-Thal iPSC-derived HSCs in transplantation.
URL [本文引用: 1]
β-thalassemia, one of the most common genetic diseases worldwide, is caused by mutations in the human hemoglobin beta (HBB) gene. Creation of human induced pluripotent stem cells (iPSCs) from β-thalassemia patients could offer an approach to cure this disease. Correction of the disease-causing mutations in iPSCs could restore normal function and provide a rich source of cells for transplantation. In this study, we used the latest gene-editing tool, CRISPR/Cas9 technology, combined with the piggyBac transposon to efficiently correct the HBB mutations in patient-derived iPSCs without leaving any residual footprint. No off-target effects were detected in the corrected iPSCs, and the cells retain full pluripotency and exhibit normal karyotypes. When differentiated into erythroblasts using a monolayer culture, gene-corrected iPSCs restored expression of HBB compared to the parental iPSCs line. Our study provides an effective approach to correct HBB mutations without leaving any genetic footprint in patient-derived iPSCs, thereby demonstrating a critical step toward the future application of stem cell-based gene therapy to monogenic diseases.
URLPMID:28388605 [本文引用: 1]
In this Review, Cathomen and colleagues present the latest advances, including improvements in nuclease specificity and delivery, that will expedite the clinical translation of genome editing.
URLPMID:27406980 [本文引用: 1]
Targeted genome editing technology can correct the sickle cell disease mutation of the $\beta$-globin gene in hematopoietic stem cells. This correction supports production of red blood cells that synthesize normal hemoglobin proteins. Here, we demonstrate that Transcription Activator-Like Effector Nucleases (TALENs) and the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 nuclease system can target DNA sequences around the sickle-cell mutation in the $\beta$-globin gene for site-specific cleavage and facilitate precise correction when a homologous donor template is codelivered. Several pairs of TALENs and multiple CRISPR guide RNAs were evaluated for both on-target and off-target cleavage rates. Delivery of the CRISPR/Cas9 components to CD34+ cells led to over 18% gene modification in vitro . Additionally, we demonstrate the correction of the sickle cell disease mutation in bone marrow derived CD34+ hematopoietic stem and progenitor cells from sickle cell disease patients, leading to the production of wild-type hemoglobin. These results demonstrate correction of the sickle mutation in patient-derived CD34+ cells using CRISPR/Cas9 technology.
URLPMID:27601644 [本文引用: 1]
Abstract Hereditary persistence of fetal hemoglobin (HPFH) is a condition in some individuals who have a high level of fetal hemoglobin throughout life. Individuals with compound heterozygous 0205-thalassemia or sickle cell disease (SCD) and HPFH have milder clinical manifestations. Using RNA-guided clustered regularly interspaced short palindromic repeats-associated Cas9 (CRISPR-Cas9) genome-editing technology, we deleted, in normal hematopoietic stem and progenitor cells (HSPCs), 13 kb of the 0205-globin locus to mimic the naturally occurring Sicilian HPFH mutation. The efficiency of targeting deletion reached 31% in cells with the delivery of both upstream and downstream breakpoint guide RNA (gRNA)-guided Staphylococcus aureus Cas9 nuclease (SaCas9). The erythroid colonies differentiated from HSPCs with HPFH deletion showed significantly higher 0206-globin gene expression compared with the colonies without deletion. By T7 endonuclease 1 assay, we did not detect any off-target effects in the colonies with deletion. We propose that this strategy of using nonhomologous end joining (NHEJ) to modify the genome may provide an efficient approach toward the development of a safe autologous transplantation for patients with homozygous 0205-thalassemia and SCD.
URLPMID:26121415 [本文引用: 2]
CRISPR-Cas-mediated genome editing relies on guide RNAs that direct site-specific DNA cleavage facilitated by the Cas endonuclease. Here we report that chemical alterations to synthesized single guide RNAs (sgRNAs) enhance genome editing efficiency in human primary T cells and CD34(+) hematopoietic stem and progenitor cells. Co-delivering chemically modified sgRNAs with Cas9 mRNA or protein is an efficient RNA- or ribonucleoprotein (RNP)-based delivery method for the CRISPR-Cas system, without the toxicity associated with DNA delivery. This approach is a simple and effective way to streamline the development of genome editing with the potential to accelerate a wide array of biotechnological and therapeutic applications of the CRISPR-Cas technology.
URLPMID:28985502 [本文引用: 1]
CRISPR-Cas systems defend prokaryotes against bacteriophages and mobile genetic elements and serve as the basis for revolutionary tools for genetic engineering. Class 2 CRISPR-Cas systems use single Cas endonucleases paired with guide RNAs to cleave complementary nucleic acid targets, enabling programmable sequence-specific targeting with minimal machinery. Recent discoveries of previously unidentified CRISPR-Cas systems have uncovered a deep reservoir of potential biotechnological tools beyond the well-characterized Type II Cas9 systems. Here we review the current mechanistic understanding of newly discovered single-protein Cas endonucleases. Comparison of these Cas effectors reveals substantial mechanistic diversity, underscoring the phylogenetic divergence of related CRISPR-Cas systems. This diversity has enabled further expansion of CRISPR-Cas biotechnological toolkits, with wide-ranging applications from genome editing to diagnostic tools based on various Cas endonuclease activities. These advances highlight the exciting prospects for future tools based on the continually expanding set of CRISPR-Cas systems.
URLPMID:27619566 [本文引用: 2]
The recent advent of the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-CRISPR associated protein 9 (Cas9) system for precise genome editing has revolutionized methodologies in haematology and oncology studies. CRISPR-Cas9 technology can be used to remove and correct genes or mutations, and to introduce site-specific therapeutic genes in human cells. Inherited haematological disorders represent ideal targets for CRISPR-Cas9-mediated gene therapy. Correcting disease-causing mutations could alleviate disease-related symptoms in the near future. The CRISPR-Cas9 system is also a useful tool for delineating molecular mechanisms involving haematological malignancies. Prior to the use of CRISPR-Cas9-mediated gene correction in humans, appropriate delivery systems with higher efficiency and specificity must be identified, and ethical guidelines for applying the technology with controllable safety must be established. Here, the latest applications of CRISPR-Cas9 technology in haematological disorders, current challenges and future directions are reviewed and discussed.
