优化第三代慢病毒载体稳定表达β-珠蛋白治疗β-地中海贫血的研究  

Optimization of β-globin Stable Expression Using the Third Generation Lentiviral Vector for β-thalassemia Therapy

作　　者：于榛 仝帅 白玥 钟晓松 YU Zhen;TONG Shuai;BAI Yue;ZHONG Xiao-Song(Clinical Gene and Cell Engineering Center,Beijing Shijitan Hospital,Capital Medical University,Beijing 100038,China)

机构地区：[1]首都医科大学附属北京世纪坛医院临床基因与细胞工程中心,北京100038

出　　处：《中国实验血液学杂志》2022年第3期844-850,共7页Journal of Experimental Hematology

基　　金：北京市科技计划脑科学专项(Z161100000216136)。

摘　　要：目的:优化第三代慢病毒载体HS4区实现β-珠蛋白稳定表达,为β-地中海贫血安全有效的细胞治疗提供研究依据。方法:优化第三代慢病毒表达载体HS4区构建lenti-HBB;转染小鼠红白血病细胞系MEL后,通过RT-PCR、Western-blot分析β-globin基因转录及翻译情况;进一步通过正常人脐带血及重型β-地中海贫血患者外周血CD34^(+)细胞感染lenti-HBB,经克隆形成实验、细胞涂片实验及流式细胞术检测CD34^(+)细胞是否发生红系分化;通过NSG小鼠体内实验验证lenti-HBB的安全性及有效性。结果:小鼠MEL细胞感染优化后的lenti-HBB后能稳定表达β-珠蛋白;正常人脐血及重度β-地中海贫血患者外周血CD34^(+)细胞感染优化后的lenti-HBB后可分化为红细胞;人CD34^(+)细胞感染优化后的lenti-HBB移植NSG小鼠3.5个月后表达β-珠蛋白。结论:通过优化第三代慢病毒载体HS4区稳定表达CD34^(+)细胞β-珠蛋白,对重型β-地中海贫血及其他β-珠蛋白异常疾病是安全有效的。Objective:To provide a research basis for a safe and effective cell therapy forβ-thalassemia through optimization of HS4 region of the third generation lentiviral vector for stable expression ofβ-globin.Methods:The humanβ-globin HS4 region in the third generation lentiviral expression vector was optimized to construct the lenti-HBB,and the transcription and translation ofβ-globin gene were analyzed by RT-PCR and Western blot after the transduction of lenti-HBB in MEL cell line.Furthermore,the erythroid differentiation of CD34^(+)cells which were transduced lentiviral virus carrying humanβ-globin from normal human umbilical cord blood cells and peripheral blood cells of patients withβ-thalassemia major were confirmed by colony formation assay,cell smear assay and flow cytometry.The safety and effectiveness of the optimized lenti-HBB were verified by NSG mouse in vivo test.Results:The humanβ-globin was expressed stably in the MEL cells,and CD34^(+)cells from health umbilical cord blood as well as PBMC from patient withβ-thalassemia major transduced with lenti-HBB could be differentiated to mature red blood cells.Theβ-globin expression and differentiation in CD34^(+)cells were demonstrated successfully in the NSG mouse for about 3.5 months after post-transplant.Conclusion:Stableβ-globin expression through the optimization of HS4 from CD34^(+)in the third generation lentiviral vector is safe and effective for patients with severeβ-thalassemia and otherβ-globin abnormal diseases.

关 键 词：Β-珠蛋白 慢病毒 Β-地中海贫血 基因治疗 

分 类 号：R556.61[医药卫生—血液循环系统疾病]