基于CRISPR/Cas9技术的β-地中海贫血和血友病基因治疗研究进展

doi:10.16288/j.yczz.20-110

遗传 ›› 2020, Vol. 42 ›› Issue (10): 949-964.doi: 10.16288/j.yczz.20-110

基于CRISPR/Cas9技术的β-地中海贫血和血友病基因治疗研究进展

鲍莉雯¹(), 周一叶², 曾凡一^1,²()

^1. 上海交通大学附属儿童医院,上海市儿童医院,上海交通大学医学遗传研究所,国家卫生健康委员会医学胚胎分子生物学重点实验室,上海市胚胎与生殖工程重点实验室,上海 200040
^2. 上海交通大学基础医学院组织胚胎学与遗传发育学系,上海 200025

收稿日期:2020-04-21 修回日期:2020-08-25 出版日期:2020-10-20 发布日期:2020-09-15
通讯作者: 曾凡一 E-mail:blwbaoliwen@163.com;fzeng@vip.163.com
作者简介:鲍莉雯,硕士研究生,专业方向：生物学。E-mail: blwbaoliwen@163.com
基金资助:
国家重点研发计划编号(2016YFC1000503);上海市重中之重重点学科项目编号(2017ZZ02019);上海市临床重点专科项目编号(shslczdzk05705)

Advances in gene therapy for β-thalassemia and hemophilia based on the CRISPR/Cas9 technology

Liwen Bao¹(), Yiye Zhou², Fanyi Zeng^1,²()

^1. Shanghai Key Laboratory of Embryo and Reproduction Engineering, Key Laboratory of Embryo Molecular Biology of National Health Commission, Shanghai Institute of Medical Genetics, Shanghai Children’s Hospital, Shanghai Jiao Tong University, Shanghai 200040, China;
^2. Department of Histoembryology, Genetics & Development, Shanghai Jiao Tong University College of Basic Medical Sciences, Shanghai 200025, China;

Received:2020-04-21 Revised:2020-08-25 Online:2020-10-20 Published:2020-09-15
Contact: Zeng Fanyi E-mail:blwbaoliwen@163.com;fzeng@vip.163.com
Supported by:
Supported by the National Key Research and Development Program of China No(2016YFC1000503);Shanghai Key Disciplines Program No(2017ZZ02019);Key Clinical Specialty Projects in Shanghai No(shslczdzk05705)

摘要/Abstract

摘要：

地中海贫血和血友病是由基因异常引发的常见的遗传性血液病,难以根治且可遗传给下一代,造成严重的家庭和社会负担。基因治疗的出现为遗传性疾病提供了新的治疗方案,但自1990年第1项基因治疗临床试验被批准以来,30年间基因治疗的发展并不乐观。随着基因编辑技术的发展,尤其具有编辑效率高、操作简单、成本低等优势的第三代基因编辑技术CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/ CRISPR-associated protein 9)的发展,基因编辑介导的基因治疗越来越受到关注,有望根治地中海贫血和血友病等遗传性血液病。本文综述了近6年(2014~2020年)基于CRISPR/Cas9技术的β-地中海贫血和血友病基因治疗基础研究进展,总结了基于CRISPR/Cas9技术的基因治疗临床试验概况,并对CRISPR/Cas9技术用于基因治疗存在的问题和可能的解决方案进行探讨,以期为基于CRISPR/Cas9技术的遗传性血液病基因治疗相关研究提供参考。

关键词: CRISPR/Cas9, 基因治疗, 遗传性血液病, 临床试验

Abstract:

Thalassemia and hemophilia are common inherited blood disorders caused by genetic abnormalities. These diseases are difficult to cure and can be inherited to the next generation, causing severe family and social burden. The emergence of gene therapy provides a new treatment for genetic diseases. However, since its first clinical trial in 1990, the development of gene therapy has not been as optimistic in the past three decades as one could hope. The development of gene-editing technology, particularly the third generation gene-editing technology CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9), has given hope in such therapeutic approach for having advantages in high editing efficiency, simple operation, and low cost. Gene editing-mediated gene therapy has thus received increasing attention from the biomedical community. It has shown promises for the treatment of inherited blood disorders, such as thalassemia and hemophilia. This paper reviews the fundamental research progress of gene therapy for β-thalassemia and hemophilia based on CRISPR/Cas9 technology in the past six years. It also summarizes the CRISPR/Cas9-based clinical trials of gene therapy. The problems and possible solutions to this technology for gene therapy are also discussed, thereby providing a reference for the research on gene therapy of inherited blood disorders based on CRISPR/Cas9 technology.

Key words: CRISPR/Cas9, gene therapy, inherited blood disorders, clinical trials

鲍莉雯, 周一叶, 曾凡一. 基于CRISPR/Cas9技术的β-地中海贫血和血友病基因治疗研究进展[J]. 遗传, 2020, 42(10): 949-964.

Liwen Bao, Yiye Zhou, Fanyi Zeng. Advances in gene therapy for β-thalassemia and hemophilia based on the CRISPR/Cas9 technology[J]. Hereditas(Beijing), 2020, 42(10): 949-964.

图/表 9

表1

图1

图2

表2

表3

图3

表4

表5

基于CRISPR/Cas9技术的基因治疗临床试验"

NCT编号^t	国家	治疗疾病	治疗途径	编辑细胞	编辑方式	试验阶段	状态
NCT03745287	美国、比利时、加拿大、德国和意大利	镰状细胞贫血	体外	自体CD34⁺ HSPC^a	破坏BCL11A^b的红系特异增强子	临床I/II期	招募中
NCT03655678	美国、加拿大、德国、意大利和英国	输血依赖型β-地贫(非β⁰/β⁰)	体外	自体CD34⁺ HSPC	破坏BCL11A的红系特异增强子	临床I/II期	招募中
NCT04205435	中国	重型β地贫	体外	自体HSC	修复HBB IVS2-654(C>T)突变	临床I/II期	未开始招募
NCT03728322	N/A^s	地贫	体外	自体HSC	HBB基因突变的原位修复	早期I期临床	未开始招募
NCT04037566	中国	CD19⁺白血病/淋巴瘤	体外	自体T细胞	敲除靶向CD19^c的CAR-T^d细胞内源性HPK1^e	临床I期	招募中
NCT04035434	美国、澳大利亚和德国	CD19⁺ B细胞恶性肿瘤	体外	异体T细胞	敲入靶向CD19的CAR并敲除TCR^f和MHC I^g	临床I/II期	招募中
NCT03166878	中国	CD19⁺ B细胞白血病/淋巴瘤	体外	异体T细胞	敲除靶向CD19的CAR-T细胞内源性TCR和B2M^h	临床I/II期	招募中
NCT03398967	中国	复发难治性B细胞恶性肿瘤	体外	异体T细胞	N/A	临床I/II期	招募中
NCT03690011	美国	T细胞白血病或淋巴瘤	体外	自体T细胞	敲除靶向CD7ⁱ和CD28^j的CAR-T细胞内源性CD7	临床I期	未开始招募
NCT04244656	美国、澳大利亚和西班牙	多发性骨髓瘤	体外	异体T细胞	敲入靶向BCMA^k的CAR并敲除TCR和MHC I	临床I期	招募中
NCT03399448	美国	多发性骨髓瘤黑色素瘤滑膜肉瘤黏液样/圆形细胞脂肪肉瘤	体外	自体T细胞	敲除靶向 NY-ESO-1^l阳性癌细胞的T细胞内源性TCR和 PDCD1^m	临床I期	终止
NCT03044743	中国	EBVⁿ阳性IV期胃癌 EBV阳性鼻咽癌 EBV阳性淋巴瘤	体外	自体T细胞	敲除T细胞内源性PDCD1	临床I/II期	招募中
NCT03747965	中国	间皮素阳性实体瘤	体外	靶向间皮素的CAR-T细胞	敲除靶向间皮素的CAR-T细胞内源性 PDCD1	临床I期	招募中
NCT03545815	中国	间皮素阳性实体瘤	体外	靶向间皮素的CAR-T细胞	敲除靶向间皮素的CAR-T细胞内源性 PDCD1和TCR	临床I期	招募中
NCT04426669	美国	转移性胃肠上皮癌	体外	自体肿瘤浸润性淋巴细胞	破坏肿瘤浸润性淋巴细胞CISH^o基因	临床I/II期	招募中
NCT03081715	中国	食管癌	体外	自体T细胞	敲除T细胞内源性PDCD1	N/A	完成
NCT02863913	中国	浸润性膀胱癌IV期	体外	自体T细胞	敲除T细胞内源性PDCD1	临床I期	撤回
NCT04438083	澳大利亚	肾细胞癌	体外	异体T细胞	敲入靶向CD70的CAR并敲除TCR和MHC I	临床I期	招募中
NCT02867332	N/A	转移性肾细胞癌	体外	自体T细胞	敲除T细胞内源性PDCD1	临床I期	撤回
NCT编号^t	国家	治疗疾病	治疗途径	编辑细胞	编辑方式	试验阶段	状态
NCT02867345	中国	激素难治性前列腺癌	体外	自体T细胞	敲除T细胞内源性PDCD1	N/A	撤回
NCT02793856	中国	转移性非小细胞肺癌	体外	自体T细胞	敲除T细胞内源性PDCD1	临床I期	未开始招募
NCT04417764	中国	晚期肝癌	体外	自体T细胞	敲除T细胞内源性PDCD1	临床I期	招募中
NCT03057912	中国	人乳头瘤病毒相关恶性肿瘤	阴道栓剂	人乳头瘤病毒	破坏人乳头瘤病毒E6/E7基因	临床I期	未知
NCT03164135	中国	HIV-1感染合并有血液肿瘤	体外	异体CD34⁺ HSPC	敲除CCR5^p基因	N/A	招募中
NCT03855631	法国	歌舞伎综合征1	细胞水平研究	自体成纤维细胞	表观修饰提升野生KMT2D^q表达	N/A	未开始招募
NCT03872479	美国	Leber先天性黑蒙症10型	体内(视网膜下注射)	N/A	破坏CEP290 IVS26 A>G突变^r	临床I/II期	招募中

表5

表6

参考文献 78

[1]	Taher AT, Weatherall DJ, Cappellini MD . Thalassaemia. Lancet, 2018,391(10116):155-167. doi: 10.1016/S0140-6736(17)31822-6 pmid: 28774421
[2]	Farashi S, Harteveld CL . Molecular basis of α-thalassemia. Blood Cells Mol Dis, 2018,70:43-53. doi: 10.1016/j.bcmd.2017.09.004 pmid: 29032940
[3]	Zeng YT, Huang SZ . Disorders of haemoglobin in China. J Med Genet, 1987,24(10):578-583. doi: 10.1136/jmg.24.10.578 pmid: 3500312
[4]	Liu JW, Hong T, Qin X, Liang YM, Zhang P . Recent advance on genome editing for therapy of β-hemoglobinopathies. Hereditas(Beijing), 2018,40(2):95-103.
	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, 2018,40(2):95-103.
[5]	Santagostino E, Fasulo MR . Hemophilia A and hemophilia B: different types of diseases? Semin Thromb Hemost, 2013,39(7):697-701. doi: 10.1055/s-0033-1353996 pmid: 24014073
[6]	Blaese RM, Culver KW, Miller AD, Carter CS, Fleisher T, Clerici M, Shearer G, Chang L, Chiang Y, Tolstoshev P, Greenblatt JJ, Rosenberg SA, Klein H, Berger M, Mullen CA, Ramsey WJ, Muul L, Morgan RA, Anderson WF . T lymphocyte-directed gene therapy for ADA- SCID: initial trial results after 4 years. Science, 1995,270(5235):475-480. doi: 10.1126/science.270.5235.475 pmid: 7570001
[7]	Niu XR, Yin SM, Chen X, Shao TT, Li DL . Gene editing technology and its recent progress in disease therapy. Hereditas(Beijing), 2019,41(7):582-598.
	牛煦然, 尹树明, 陈曦, 邵婷婷, 李大力 . 基因编辑技术及其在疾病治疗中的研究进展. 遗传, 2019,41(7):582-598.
[8]	Nathwani AC, Davidoff AM, Tuddenham EGD . Advances in gene therapy for hemophilia. Hum Gene Ther, 2017,28(11):1004-1012. doi: 10.1089/hum.2017.167 pmid: 28835123
[9]	Peyvandi F, Garagiola I . Clinical advances in gene therapy updates on clinical trials of gene therapy in haemophilia. Haemophilia, 2019,25(5):738-746. doi: 10.1111/hae.13816 pmid: 31282050
[10]	Zeng YT, Huang SZ, Ren ZR, Lu ZH, Zeng FY, Schechter AN, Rodgers GP . Hydroxyurea therapy in beta-thalassaemia intermedia: improvement in haematological parameters due to enhanced beta-globin synthesis. Br J Haematol, 1995,90(3):557-563. pmid: 7646994
[11]	Xie SY, Li W, Ren ZR, Huang SZ, Zeng FY, Zeng YT . Correction of β654-thalassaemia mice using direct intravenous injection of siRNA and antisense RNA vectors. Int J Hematol, 2011,93(3):301-310. doi: 10.1007/s12185-010-0727-1
[12]	Xie SY, Ren ZR, Zhang JZ, Guo XB, Wang QX, Wang S, Lin D, Gong XL, Li W, Huang SZ, Zeng FY, Zeng YT . Restoration of the balanced alpha/beta-globin gene expression in beta654-thalassemia mice using combined RNAi and antisense RNA approach. Hum Mol Genet, 2007,16(21):2616-2625. doi: 10.1093/hmg/ddm218 pmid: 17716993
[13]	Davis R, Gurumurthy A, Hossain MA, Gunn EM, Bungert J . Engineering globin gene expression. Mol Ther Methods Clin Dev, 2018,12:102-110. doi: 10.1016/j.omtm.2018.12.004 pmid: 30603654
[14]	Elalfy MS, Adly AAM, Ismail EA, Elhenawy YI, Elghamry IR . Therapeutic superiority and safety of combined hydroxyurea with recombinant human erythropoietin over hydroxyurea in young β-thalassemia intermedia patients. Eur J Haematol, 2013,91(6):522-533. doi: 10.1111/ejh.12182
[15]	Xie F, Ye L, Chang JC, Beyer AI, Wang JM, Muench MO, Kan YW . Seamless gene correction of β-thalassemia mutations in patient-specific iPSCs using CRISPR/Cas9 and piggyBac. Genome Res, 2014,24(9):1526-1533.
[16]	Ren YX, Xiao RD, Lou XM, Fang XD . Research advance and application in the gene therapy of gene editing technologies. Hereditas (Beijing), 2019,41(1):18-27.
	任云晓, 肖茹丹, 娄晓敏, 方向东 . 基因编辑技术及其在基因治疗中的应用. 遗传, 2019,41(1):18-27.
[17]	Garneau JE, Dupuis MÈ, Villion M, Romero DA, Barrangou R, Boyaval P, Fremaux C, Horvath P, Magadán AH, Moineau S . The CRISPR/Cas bacterial immune system cleaves bacteriophage and plasmid DNA. Nature, 2010,468(7320):67-71. doi: 10.1038/nature09523 pmid: 21048762
[18]	Deltcheva E, Chylinski K, Sharma CM, Gonzales K, Chao YJ, Pirzada ZA, Eckert MR, Vogel J, Charpentier E . CRISPR RNA maturation by trans-encoded small RNA and host factor RNase III. Nature, 2011,471(7340):602-607. doi: 10.1038/nature09886 pmid: 21455174
[19]	Jinek M, Chylinski K, Fonfara I, Haue M, Doudna JA, Charpentier E . A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science, 2012,337(6096):816-821. doi: 10.1126/science.1225829 pmid: 22745249
[20]	Mali P, Yang LH, Esvelt KM, Aach J, Guell M , DiCarlo JE, Norville JE, Church GM. RNA-guided human genome engineering via Cas9. Science, 2013,339(6121):823-826.
[21]	Zhang JH, Adikaram P, Pandey M, Genis A, Simonds WF . Optimization of genome editing through CRISPR-Cas9 engineering. Bioengineered, 2016,7(3):166-174. doi: 10.1080/21655979.2016.1189039 pmid: 27340770
[22]	Chang HHY, Pannunzio NR, Adachi N, Lieber MR . Non-homologous DNA end joining and alternative pathways to double-strand break repair. Nat Rev Mol Cell Biol, 2017,18(8):495-506. pmid: 28512351
[23]	Li GL, Zhong CL, Mo JX, Quan R, Wu ZF, Li ZC, Yang HQ, Zhang XW . Advances in site-specific integration of transgene in animal genome. Hereditas (Beijing), 2017,39(2):98-109.
	李国玲, 钟翠丽, 莫健新, 全绒, 吴珍芳, 李紫聪, 杨化强, 张献伟 . 动物基因组定点整合转基因技术研究进展. 遗传, 2017,39(2):98-109.
[24]	Zong Y, Gao CX . Progress on base editing systems. Hereditas (Beijing), 2019,41(9):777-800.
	宗媛, 高彩霞 . 碱基编辑系统研究进展. 遗传, 2019,41(9):777-800.
[25]	Liang PP, Sun HW, Sun Y, Zhang XY, Xie XW, Zhang JR, Zhang Z, Chen YX, Ding CH, Xiong YY, Ma WB, Liu D, Huang JJ, Zhou SY . Effective gene editing by high-fidelity base editor 2 in mouse zygotes. Protein Cell, 2017,8(8):601-611. doi: 10.1007/s13238-017-0418-2 pmid: 28585179
[26]	Kim K, Ryu SM, Kim ST, Baek G, Kim D, Lim K, Chung E, Kim S, Kim JS . Highly efficient RNA-guided base editing in mouse embryos. Nat Biotechnol, 2017,35(5):435-437. doi: 10.1038/nbt.3816 pmid: 28244995
[27]	Lai KT, Huang GF, Su L, He YY . The prevalence of thalassemia in mainland China: evidence from epidemiological surveys. Sci Rep, 2017,7(1):920. doi: 10.1038/s41598-017-00967-2 pmid: 28424478
[28]	Niu XH, He WY, Song B, Ou ZH, Fan D, Chen YC, Fan Y, Sun XF . Combining single strand oligodeoxynucleotides and CRISPR/Cas9 to correct gene mutations in β-thalassemia-induced pluripotent stem cells. J Biol Chem, 2016,291(32):16576-16585. doi: 10.1074/jbc.M116.719237
[29]	Song B, Fan Y, He WY, Zhu DT, Niu XH, Wang D, Ou ZH, Luo M, Sun XF . Improved hematopoietic differentiation efficiency of gene-corrected beta-thalassemia induced pluripotent stem cells by CRISPR/Cas9 system. Stem Cells Dev, 2015,24(9):1053-1065. doi: 10.1089/scd.2014.0347 pmid: 25517294
[30]	Liu YL, Yang Y, Kang XJ, Lin B, Yu Q, Song B, Gao G, Chen YY, Sun XF, Li XP, Bu L, Fan Y . One-step biallelic and scarless correction of a β-thalassemia mutation in patient-specific iPSCs without drug selection. Mol Ther Nucleic Acids, 2017,6:57-67. doi: 10.1016/j.omtn.2016.11.010 pmid: 28325300
[31]	Park SH, Lee CM, Dever DP, Davis TH, Camarena J, Srifa W, Zhang YK, Paikari A, Chang AK, Porteus MH, Sheehan VA, Bao G . Highly efficient editing of the β-globin gene in patient-derived hematopoietic stem and progenitor cells to treat sickle cell disease. Nucleic Acids Res, 2019,47(15):7955-7972. doi: 10.1093/nar/gkz475 pmid: 31147717
[32]	Wattanapanitch M, Damkham N, Potirat P, Trakarnsanga K, Janan M, U-pratya Y, Kheolamai P, Klincumhom N, Issaragrisil S,. One-step genetic correction of hemoglobin E/beta-thalassemia patient-derived iPSCs by the CRISPR/Cas9 system. Stem Cell Res Ther, 2018,9(1):46. doi: 10.1186/s13287-018-0779-3
[33]	Antony JS, Latifi N, Haque AKMA, Lamsfus-Calle A, Daniel-Moreno A, Graeter S, Baskaran P, Weinmann P, Mezger M, Handgretinger R, Kormann MSD . Gene correction of HBB mutations in CD34 + hematopoietic stem cells using Cas9 mRNA and ssODN donors . Mol Cell Pediatr, 2018,5(1):9. doi: 10.1186/s40348-018-0086-1 pmid: 30430274
[34]	Fang YD, Cheng Y, Lu D, Gong XL, Yang GH, Gong ZJ, Zhu YW, Sang X, Fan SY, Zhang JZ, Zeng FY . Treatment of β ⁶⁵⁴ -thalassaemia by TALENs in a mouse model . Cell Prolif, 2018,51(6):e12491. doi: 10.1111/cpr.12491 pmid: 30070404
[35]	Xu SQ, Luk K, Yao QM, Shen AH, Zeng J, Wu YX, Luo HY, Brendel C, Pinello L, Chui DHK, Wolfe SA, Bauer DE . Editing aberrant splice sites efficiently restores β-globin expression in β-thalassemia. Blood, 2019,133(21):2255-2262. doi: 10.1182/blood-2019-01-895094 pmid: 30704988
[36]	Park CY, Kim DH, Son JS, Sung JJ, Lee J, Bae S, Kim JH, Kim DW, Kim JS . Functional correction of large factor VIII gene chromosomal inversions in hemophilia A patient-derived iPSCs using CRISPR-Cas9. Cell Stem Cell, 2015,17(2):213-220. doi: 10.1016/j.stem.2015.07.001 pmid: 26212079
[37]	Peng Z, Zhou WC, Fu WQ, Du RQ, Jin L, Zhang F . Correlation between frequency of non-allelic homologous recombination and homology properties: evidence from homology-mediated CNV mutations in the human genome. Hum Mol Genet, 2015,24(5):1225-1233. doi: 10.1093/hmg/ddu533 pmid: 25324539
[38]	Sasaki M, Lange J, Keeney S . Genome destabilization by homologous recombination in the germ line. Nat Rev Mol Cell Biol, 2010,11(3):182-195. doi: 10.1038/nrm2849 pmid: 20164840
[39]	Park CY, Kim J, Kweon J, Son JS, Lee JS, Yoo JE, Cho SR, Kim JH, Kim JS, Kim DW . Targeted inversion and reversion of the blood coagulation factor 8 gene in human iPS cells using TALENs. Proc Natl Acad Sci USA, 2014,111(25):9253-9258. doi: 10.1073/pnas.1323941111
[40]	George CM, Alani E . Multiple cellular mechanisms prevent chromosomal rearrangements involving repetitive DNA. Crit Rev Biochem Mol Biol, 2012,47(3):297-313. doi: 10.3109/10409238.2012.675644 pmid: 22494239
[41]	Zeng J, Wu YX, Ren CY, Bonanno J, Shen AH, Shea D, Gehrke JM, Clement K, Luk K, Yao QM, Kim R, Wolfe SA, Manis JP, Pinello L, Joung JK, Bauer DE . Therapeutic base editing of human hematopoietic stem cells. Nat Med, 2020,26(4):535-541. doi: 10.1038/s41591-020-0790-y pmid: 32284612
[42]	Zhou CY, Sun YD, Yan R, Liu YJ, Zuo EW, Gu C, Han LX, Wei Y, Hu XD, Zeng R, Li YX, Zhou HB, Guo F, Yang H . Off-target RNA mutation induced by DNA base editing and its elimination by mutagenesis. Nature, 2019,571(7764):275-278. doi: 10.1038/s41586-019-1314-0 pmid: 31181567
[43]	Thein SL . Molecular basis of β thalassemia and potential therapeutic targets. Blood Cells Mol Dis, 2018,70:54-65. doi: 10.1016/j.bcmd.2017.06.001 pmid: 28651846
[44]	Mettananda S, Fisher CA, Hay D, Badat M, Quek L, Clark K, Hublitz P, Downes D, Kerry J, Gosden M, Telenius J, Sloane-Stanley JA, Faustino P, Coelho A, Doondeea J, Usukhbayar B, Sopp P, Sharpe JA, Hughes JR, Vyas P, Gibbons RJ, Higgs DR . Editing an α-globin enhancer in primary human hematopoietic stem cells as a treatment for β-thalassemia. Nat Commun, 2017,8(1):424. doi: 10.1038/s41467-017-00479-7 pmid: 28871148
[45]	Wu YX, Zeng J, Roscoe BP, Liu PP, Yao QM, Lazzarotto CR, Clement K, Cole MA, Luk K, Baricordi C, Shen AH, Ren CY, Esrick EB, Manis JP, Dorfman DM, Williams DA, Biffi A, Brugnara C, Biasco L, Brendel C, Pinello L, Tsai SQ, Wolfe SA, Bauer DE . Highly efficient therapeutic gene editing of human hematopoietic stem cells. Nat Med, 2019,25(5):776-783. doi: 10.1038/s41591-019-0401-y pmid: 30911135
[46]	Martyn GE, Wienert B, Yang L, Shah M, Norton LJ, Burdach J, Kurita R, Nakamura Y, Pearson RCM, Funnell APW, Quinlan KGR, Crossley M . Natural regulatory mutations elevate the fetal globin gene via disruption of BCL11A or ZBTB7A binding. Nat Genet, 2018,50(4):498-503. doi: 10.1038/s41588-018-0085-0 pmid: 29610478
[47]	Martyn GE, Wienert B, Kurita R, Nakamura Y, Quinlan KGR, Crossley M . A natural regulatory mutation in the proximal promoter elevates fetal globin expression by creating a de novo GATA1 site. Blood, 2019,133(8):852-856. doi: 10.1182/blood-2018-07-863951 pmid: 30617196
[48]	Antoniani C, Meneghini V, Lattanzi A, Felix T, Romano O, Magrin E, Weber L, Pavani G, Hoss SE, Kurita R, Nakamura Y, Cradick TJ, Lundberg AS, Porteus M, Amendola M, Nemer WE, Cavazzana M, Mavilio F, Miccio A . Induction of fetal hemoglobin synthesis by CRISPR/Cas9-mediated editing of the human β-globin locus. Blood, 2018,131(17):1960-1973. doi: 10.1182/blood-2017-10-811505 pmid: 29519807
[49]	Canver MC, Smith EC, Sher F, Pinello L, Sanjana NE, Shalem O, Chen DD, Schupp PG, Vinjamur DS, Garcia SP, Luc S, Kurita R, Nakamura Y, Fujiwara Y, Maeda T, Yuan GC, Zhang F, Orkin SH, Bauer DE . BCL11A enhancer dissection by Cas9-mediated in situ saturating mutagenesis. Nature, 2015,527(7577):192-197. doi: 10.1038/nature15521 pmid: 26375006
[50]	Liu PT, Keller JR, Ortiz M, Tessarollo L, Rachel RA, Nakamura T, Jenkins NA, Copeland NG . Bcl11a is essential for normal lymphoid development. Nat Immunol, 2003,4(6):525-532. doi: 10.1038/ni925 pmid: 12717432
[51]	Li J, Lai YR, Shi LL . BCL11A down-regulation induces γ-globin in human β-thalassemia major erythroid cells. Hemoglobin, 2018,42(4):225-230. doi: 10.1080/03630269.2018.1515774 pmid: 30821197
[52]	Wang LR, Li LX, Ma YL, Hu HD, Li Q, Yang Y, Liu WB, Yin SM, Li W, Fu B, Kurita R, Nakamura Y, Liu M, Lai YR, Li DL . Reactivation of γ-globin expression through Cas9 or base editor to treat β-hemoglobinopathies. Cell Res, 2020,30(3):276-278. doi: 10.1038/s41422-019-0267-z pmid: 31911671
[53]	Grevet JD, Lan XJ, Hamagami N, Edwards CR, Sankaranarayanan L, Ji XJ, Bhardwaj SK, Face CJ, Posocco DF, Abdulmalik O, Keller CA, Giardine B, Sidoli S, Garcia BA, Chou ST, Liebhaber SA, Hardison RC, Shi JW, Blobel GA . Domain-focused CRISPR screen identifies HRI as a fetal hemoglobin regulator in human erythroid cells. Science, 2018,361(6399):285-290. doi: 10.1126/science.aao0932 pmid: 30026227
[54]	Thompson AA, Walters MC, Kwiatkowski J, Rasko JEJ, Ribeil JA, Hongeng S, Magrin E, Schiller GJ, Payen E, Semeraro M, Moshous D, Lefrere F, Puy H, Bourget P, Magnani A, Caccavelli L, Diana JS, Suarez F, Monpoux F, Brousse V, Poirot C, Brouzes C, Meritet JF, Pondarré C, Beuzard Y, Chrétien S, Lefebvre T, Teachey DT, Anurathapan U, Ho PJ, von Kalle C, Kletzel M, Vichinsky E, Soni S, Veres G, Negre O, Ross RW, Davidson D, Petrusich A, Sandler L, Asmal M, Hermine O, Montalembert MD, Hacein-Bey-Abina S, Blanche S, Leboulch P, Cavazzana M,. Gene therapy in patients with transfusion-dependent β-thalassemia. N Engl J Med, 2018,378(16):1479-1493. doi: 10.1056/NEJMoa1705342 pmid: 29669226
[55]	Chen YH, Keiser MS, Davidson BL . Viral vectors for gene transfer. Curr Protoc Mouse Biol, 2018,8(4):e58. doi: 10.1002/cpmo.58 pmid: 30485696
[56]	Cunningham SC, Dane AP, Spinoulas A, Alexander IE . Gene delivery to the juvenile mouse liver using AAV2/8 vectors. Mol Ther, 2008,16(6):1081-1088. doi: 10.1038/mt.2008.72 pmid: 18414478
[57]	George LA, Sullivan SK, Giermasz A, Rasko JEJ, Samelson-Jones BJ, Ducore J, Cuker A, Sullivan LM, Majumdar S, Teitel J, McGuinn CE, Ragni MV, Luk AY, Hui D, Wright JF, Chen YF, Liu Y, Wachtel K, Winters A, Tiefenbacher S, Arruda VR, van der Loo JCM, Zelenaia O, Takefman D, Carr ME, Couto LB, Anguela XM, High KA,. Hemophilia B gene therapy with a high-specific-activity factor IX variant. N Engl J Med, 2017,377(23):2215-2227. doi: 10.1056/NEJMoa1708538 pmid: 29211678
[58]	Wang LL, Yang Y, Breton CA, White J, Zhang J, Che Y, Saveliev A, McMenamin D, He ZN, Latshaw C, Li MY, Wilson JM, . CRISPR/Cas9-mediated in vivo gene targeting corrects hemostasis in newborn and adult factor IX-knockout mice. Blood, 2019,133(26):2745-2752. doi: 10.1182/blood.2019000790 pmid: 30975639
[59]	Baliou S, Adamaki M, Kyriakopoulos AM, Spandidos DA, Panayiotidis M, Christodoulou I, Zoumpourlis V . CRISPR therapeutic tools for complex genetic disorders and cancer (Review). Int J Oncol, 2018,53(2):443-468. doi: 10.3892/ijo.2018.4434 pmid: 29901119
[60]	Cyranoski D . CRISPR gene-editing tested in a person for the first time. Nature, 2016,539(7630):479. pmid: 27882996
[61]	Lu Y, Xue JX, Deng T, Zhou XJ, Yu K, Deng L, Huang MJ, Yi X, Liang MZ, Wang Y, Shen HG, Tong RZ, Wang WB, Li L, Song J, Li J, Su XX, Ding ZY, Gong YL, Zhu J, Wang YS, Zou BW, Zhang Y, Li YY, Zhou L, Liu YM, Yu M, Wang YQ, Zhang XW, Yin LM, Xia XF, Zeng Y, Zhou Q, Ying BW, Chen C, Wei YQ, Li WM, Mok T . Safety and feasibility of CRISPR-edited T cells in patients with refractory non-small-cell lung cancer. Nat Med, 2020,26(5):732-740. doi: 10.1038/s41591-020-0840-5 pmid: 32341578
[62]	Stadtmauer EA, Fraietta JA, Davis MM, Cohen AD, Weber KL, Lancaster E, Mangan PA, Kulikovskaya I, Gupta M, Chen F, Tian LF, Gonzalez VE, Xu J, Jung IY, Melenhorst JJ, Plesa G, Shea J, Matlawski T, Cervini A, Gaymon AL, Desjardins S, Lamontagne A, Salas-Mckee J, Fesnak A, Siegel DL, Levine BL, Jadlowsky JK, Young RM, Chew A, Hwang WT, Hexner EO, Carreno BM, Nobles CL, Bushman FD, Parker KR, Qi YY, Satpathy AT, Chang HY, Zhao YB, Lacey SF, June CH. CRISPR-engineered T cells in patients with refractory cancer. Science, 2020, 367(6481): eaba7365. pmid: 32108095
[63]	Xu L, Wang J, Liu YL, Xie LF, Su B, Mou DL, Wang LT, Liu TT, Wang XB, Zhang B, Zhao L, Hu LD, Ning HM, Zhang YF, Deng K, Liu LF, Lu XF, Zhang T, Xu J, Li C, Wu H, Deng HK, Chen H . CRISPR-edited stem cells in a patient with HIV and acute lymphocytic leukemia. N Engl J Med, 2019,381(13):1240-1247. doi: 10.1056/NEJMoa1817426 pmid: 31509667
[64]	Wang DW, Wang K, Cai YJ . An overview of development in gene therapeutics in China. Gene Ther, 2020,27(7-8):338-348. pmid: 32528163
[65]	Park JH, Geyer MB, Brentjens RJ . CD19-targeted CAR T-cell therapeutics for hematologic malignancies: interpreting clinical outcomes to date. Blood, 2016,127(26):3312-3320. doi: 10.1182/blood-2016-02-629063 pmid: 27207800
[66]	Fu YF, Foden JA, Khayter C, Maeder ML, Reyon D, Joung JK, Sander JD . High-frequency off-target mutagenesis induced by CRISPR-Cas nucleases in human cells. Nat Biotechnol, 2013,31(9):822-826. doi: 10.1038/nbt.2623 pmid: 23792628
[67]	Cullot G, Boutin J, Toutain J, Prat F, Pennamen P, Rooryck C, Teichmann M, Rousseau E, Lamrissi-Garcia I, Guyonnet-Duperat V, Bibeyran A, Lalanne M, Prouzet- Mauléon V, Turcq B, Ged C, Blouin JM, Richard E, Dabernat S, Moreau-Gaudry F, Bedel A . CRISPR-Cas9 genome editing induces megabase-scale chromosomal truncations. Nat Commun, 2019,10(1):1136. doi: 10.1038/s41467-019-09006-2 pmid: 30850590
[68]	Lattanzi A, Meneghini V, Pavani G, Amor F, Ramadier S, Felix T, Antoniani C, Masson C, Alibeu O, Lee C, Porteus MH, Bao G, Amendola M, Mavilio F, Miccio A . Optimization of CRISPR/Cas9 delivery to human hematopoietic stem and progenitor cells for therapeutic genomic rearrangements. Mol Ther, 2019,27(1):137-150. pmid: 30424953
[69]	Dever DP, Bak RO, Reinisch A, Camarena J, Washington G, Nicolas CE, Pavel-Dinu M, Saxena N, Wilkens AB, Mantri S, Uchida N, Hendel A, Narla A, Majeti R, Weinberg KI, Porteus MH . CRISPR/Cas9 β-globin gene targeting in human haematopoietic stem cells. Nature, 2016,539(7629):384-389. pmid: 27820943
[70]	Doench JG, Fusi N, Sullender M, Hegde M, Vaimberg EW, Donovan KF, Smith I, Tothova Z, Wilen C, Orchard R, Virgin HW, Listgarten J, Root DE . Optimized sgRNA design to maximize activity and minimize off-target effects of CRISPR-Cas9. Nat Biotechnol, 2016,34(2):184-191. doi: 10.1038/nbt.3437 pmid: 26780180
[71]	Hendel A, Bak RO, Clark JT, Kennedy AB, Ryan DE, Roy S, Steinfeld I, Lunstad BD, Kaiser RJ, Wilkens AB, Bacchetta R, Tsalenko A, Dellinger D, Bruhn L, Porteus MH . Chemically modified guide RNAs enhance CRISPR- Cas genome editing in human primary cells. Nat Biotechnol, 2015,33(9):985-989. doi: 10.1038/nbt.3290 pmid: 26121415
[72]	Vakulskas CA, Dever DP, Rettig GR, Turk R, Jacobi AM, Collingwood MA, Bode NM, McNeill MS, Yan SQ, Camarena J, Lee CM, Park SH, Wiebking V, Bak RO, Gomez-Ospina N, Pavel-Dinu M, Sun WC, Bao G, Porteus MH, Behlke MA,. A high-fidelity Cas9 mutant delivered as a ribonucleoprotein complex enables efficient gene editing in human hematopoietic stem and progenitor cells. Nat Med, 2018,24(8):1216-1224. doi: 10.1038/s41591-018-0137-0 pmid: 30082871
[73]	Shen MW, Arbab M, Hsu JY, Worstell D, Culbertson SJ, Krabbe O, Cassa CA, Liu DR, Gifford DK, Sherwood RI . Predictable and precise template-free CRISPR editing of pathogenic variants. Nature, 2018,563(7733):646-651. pmid: 30405244
[74]	Yang YY, Zhang XB, Yi L, Hou ZZ, Chen JY, Kou XC, Zhao YH, Wang H, Sun XF, Jiang CZ, Wang YX, Gao SR . Naïve induced pluripotent stem cells generated from β-thalassemia fibroblasts allow efficient gene correction with CRISPR/Cas9. Stem Cells Transl Med, 2016,5(2):267. doi: 10.5966/sctm.2015-0157erratum
[75]	Volarevic V, Markovic BS, Gazdic M, Volarevic A, Jovicic N, Arsenijevic N, Armstrong L, Djonov V, Lako M, Stojkovic M . Ethical and safety issues of stem cell-based therapy. Int J Med Sci, 2018,15(1):36-45. doi: 10.7150/ijms.21666 pmid: 29333086
[76]	Paes BCMF, Moço PD, Pereira CG, Porto GS, de Sousa Russo EM, Reis LCJ, Covas DT, Picanço-Castro V,. Ten years of iPSC: clinical potential and advances in vitro hematopoietic differentiation. Cell Biol Toxicol, 2017,33(3):233-250. doi: 10.1007/s10565-016-9377-2 pmid: 28039590
[77]	Tan YT, Ye L, Xie F, Beyer AI, Muench MO, Wang JM, Chen Z, Liu H, Chen SJ, Kan YW . Respecifying human iPSC-derived blood cells into highly engraftable hematopoietic stem and progenitor cells with a single factor. Proc Natl Acad Sci USA, 2018,115(9):2180-2185. pmid: 29386396
[78]	Uchida N, Haro-Mora JJ, Fujita A, Lee DY, Winkler T, Hsieh MM, Tisdale JF . Efficient generation of β-globin- expressing erythroid cells using stromal cell-derived induced pluripotent stem cells from patients with sickle cell disease. Stem Cells, 2017,35(3):586-596. doi: 10.1002/stem.2517 pmid: 27739611

编辑推荐

Metrics

www.chinagene.cn
备案号：京ICP备09063187号-4
总访问:,今日访问:,当前在线:

典型疾病	致病机制	突变基因	突变类型	参考文献
α-地中海贫血	α-珠蛋白链合成减少/缺失	HBA	大片段缺失	[1,2]
β-地中海贫血	β-珠蛋白链合成减少/缺失	HBB	点突变/移码突变	[1,4]
镰状细胞贫血	β-珠蛋白链结构异常	HBB	外显子点突变	[4]
血友病A	凝血因子VIII缺乏	FVIII	内含子倒位	[5]
血友病B	凝血因子IX缺乏	FIX	点突变	[5]

修复途径	模版	效率	作用时期	对细胞种类/ 状态依赖性	修复结果	适用修复突变	参考文献
同源重组修复HDR	需要	低	S期和G₂期	高	精确修复	外显子突变	[22,23]
非同源末端连接NHEJ	不需要	高	整个细胞周期	低	核苷酸插入/缺失	外显子突变	[22,23]
非同源末端连接NHEJ	不需要	高	整个细胞周期	低	不可精确修复	内含子突变	[22,23]

突变类型	主要疾病	策略	年份	编辑细胞	编辑基因	修复/编辑效率	脱靶率	编辑后体外细胞表型	参考文献
外显子点突变和移码突变	β-地贫镰状细胞贫血血友病B	HDR^a	2014	β-地贫iPSC	HBB	-28A/G: 7.8% CD41/42 -TCTT: 9.8% 双位点修复效率: 0	未检测到脱靶	HBB mRNA量升高约16倍	[15]
			2015	β-地贫iPSC	HBB	16.67%	未检测到脱靶	HBB蛋白量由0上升至正常水平的~60%	[29]
			2016	β-地贫iPSC	HBB	0.045%	未检测到脱靶	HBB蛋白量接近正常	[28]
			2016	β-地贫iPSC	HBB	na ??ve iPSC组: 57% primed iPSC组: 32%	na ??ve iPSC: 0 primed iPSC: 5%	未说明	[74]
			2017	β-地贫iPSC	HBB	双等位基因修复: 54% 单等位基因修复: 25.5%	未检测到脱靶	有HBB mRNA表达	[30]
			2018	β-地贫iPSC	HBB	2.9%	3个克隆中2个检测到HBD点突变	HBB mRNA和HBB蛋白无明显上升	[32]
			2019	镰状细胞贫血HSPC	HBB	24.5±7.6%	Cas9: 0.1%~36.7% HiFi Cas9^d: ≤0.23%	HbA水平由5.3±1%提升至25.3±13.9%	[31]
内含子点突变	β-地贫	HDR	2018	β-地贫HSC	HBB	8%	未检测到脱靶	未说明	[33]
内含子点突变	β-地贫	NHEJ^b	2019	β-地贫HSPC	HBB	indel产生率: 93%	未说明	HbA分数从36.4%上升至75.6%	[35]
内含子倒位	血友病A	NAHR^c	2015	血友病A iPSC	FVIII	3.7%	未检测到脱靶	F8 mRNA量由0上升至正常水平的50%~120%	[36]

年份	编辑细胞	编辑方式	编辑效率	脱靶率	编辑后体外细胞表型	参考文献
2015	正常人HSPC	破坏BCL11A红系特异增强子	N/A	N/A	HBG表达水平上升近4倍,分化后HbF⁺细胞占比升高近2倍	[49]
2018	HUDEP-2^a	破坏HBG近端启动子上BCL11A和ZBTB7A结合位点	N/A	N/A	mRNA水平: γ/(γ+β)%比值由不到5%上升至~70% 蛋白水平: HbF水平由0上升至58.7±9.6%	[47]
2018	SCD HSPC^b	敲除13.6kb长的可能的γ-珠蛋白基因抑制因子区域	缺失: 34.1% 倒位: 28.1%	0~4.6%	γ链/类β链的比值较对照上升近2.5倍 0%氧气条件下,镰状细胞数由~65%下降至~30%	[48]
2018	SCD CD34⁺细胞^c	敲除HRI^d	N/A	N/A	mRNA水平: HBG/(HBG+HBB)%比值由3.2%上升至24% 蛋白水平: HbF上升近4倍细胞水平: HbF⁺细胞占比升高近2倍	[53]
2019	β-地贫HSPC	破坏BCL11A红系特异增强子	平均84.4%	未检测到脱靶	HbF/(HbF+HbA+HbA₂)%比值由~30%上升至~70%	[45]
2019	HUDEP-2	破坏HBG近端启动子上BCL11A结合位点	N/A^e	N/A	mRNA水平: γ/(γ+β)%比值由~10%上升至~60% 蛋白水平: HbF/(HbF+HbA)%比值由零上升至~40%	[46]
2020	β-地贫HSPC	破坏HBG近端启动子上BCL11A结合位点	平均85%	未检测到脱靶	γ-珠蛋白的mRNA水平可达α-珠蛋白的126%	[52]

	基因编辑技术
	ZFN	TALEN	CRISPR/Cas9
识别模式	蛋白-DNA	蛋白-DNA	RNA-DNA
识别序列长度(nt)	18~36	30~40	22
操作	复杂	复杂	简单
效率	低	低	较高
脱靶率	较低	低	较高
成本	高	高	较低

基于CRISPR/Cas9技术的β-地中海贫血和血友病基因治疗研究进展

Advances in gene therapy for β-thalassemia and hemophilia based on the CRISPR/Cas9 technology

RichHTML

PDF (PC)

可视化

摘要/Abstract

引用本文

使用本文

图/表 9

参考文献 78

相关文章 15

编辑推荐

Metrics

[1]	王秉政, 张超, 张佳丽, 孙锦. 利用单转录本表达Cas9和sgRNA条件性编辑果蝇基因组[J]. 遗传, 2023, 45(7): 593-601.
[2]	刘梅珍, 王立人, 李咏梅, 马雪云, 韩红辉, 李大力. 利用CRISPR/Cas9技术构建基因编辑大鼠模型[J]. 遗传, 2023, 45(1): 78-87.
[3]	张潇筠, 徐坤, 沈俊岑, 穆璐, 钱泓润, 崔婕妤, 马宝霞, 陈知龙, 张智英, 魏泽辉. 一种新型提高HDR效率的CRISPR/Cas9-Gal4BD供体适配基因编辑系统[J]. 遗传, 2022, 44(8): 708-719.
[4]	张充, 魏子璇, 王敏, 陈瑶生, 何祖勇. 利用CRISPR/Cas9在人类黑色素瘤细胞中编辑MC1R与功能分析[J]. 遗传, 2022, 44(7): 581-590.
[5]	刘尧, 周先辉, 黄舒泓, 王小龙. 引导编辑：突破碱基编辑类型的新技术[J]. 遗传, 2022, 44(11): 993-1008.
[6]	韩玉婷, 许博文, 李羽童, 卢心怡, 董习之, 邱雨浩, 车沁耘, 朱芮葆, 郑丽, 李孝宸, 司绪, 倪建泉. 模式动物果蝇的基因调控前沿技术[J]. 遗传, 2022, 44(1): 3-14.
[7]	杨光武, 田嫄. 果蝇F-box基因Ppa促进脂肪储存[J]. 遗传, 2021, 43(6): 615-622.
[8]	彭定威, 李瑞强, 曾武, 王敏, 石翾, 曾检华, 刘小红, 陈瑶生, 何祖勇. 编辑MSTN半胱氨酸节基元促进两广小花猪肌肉生长[J]. 遗传, 2021, 43(3): 261-270.
[9]	王娜, 甲芝莲, 吴强. RFX5调控原钙粘蛋白α基因簇的表达[J]. 遗传, 2020, 42(8): 760-774.
[10]	李国玲, 杨善欣, 吴珍芳, 张献伟. 提高CRISPR/Cas9介导的动物基因组精确插入效率研究进展[J]. 遗传, 2020, 42(7): 641-656.
[11]	陈赢男, 陆静. CRISPR/Cas9系统在林木基因编辑中的应用[J]. 遗传, 2020, 42(7): 657-668.
[12]	刘思远, 易国强, 唐中林, 陈斌. 基于CRISPR/Cas9系统在全基因组范围内筛选功能基因及调控元件研究进展[J]. 遗传, 2020, 42(5): 435-443.
[13]	林珉婷, 赖璐璐, 赵淼, 林必玮, 姚香平. 利用CRISPR/Cas9 AAV系统构建纹状体Slc20a2基因敲除小鼠模型[J]. 遗传, 2020, 42(10): 1017-1027.
[14]	刘沛峰, 吴强. CRISPR/Cas9基因编辑在三维基因组研究中的应用[J]. 遗传, 2020, 42(1): 18-31.
[15]	牛煦然,尹树明,陈曦,邵婷婷,李大力. 基因编辑技术及其在疾病治疗中的研究进展[J]. 遗传, 2019, 41(7): 582-598.