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

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

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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)

Abstract

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

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.

Figures/Tables 9

Table 1

Fig. 1

Fig. 2

Table 2

Table 3

Fig. 3

Table 4

Table 5

Clinical trials of gene therapy based on CRISPR/Cas9 technology"

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

Table 5

Table 6

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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

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

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