【摘要】 目的探讨慢病毒载体中红细胞特异性基因调控元件的优化对小鼠红白血病(murine erythroleu-kemia,MEL)细胞向红系终端分化期间组织特异性表达重组蛋白的影响。方法改变红细胞特异性β-珠蛋白启动子编码长度、改变β-珠蛋白基因位点控制区DNaseⅠ高敏位点(hypersensitive site,HS)的组合、删除β-珠蛋白3’端增强子或β-珠蛋白内含子IVS2,由此构建一系列由不同缺失组合的基因调控元件控制目的基因(如人凝血因子Ⅸ)表达的慢病毒载体,包装成重组慢病毒,测定病毒滴度,并分别感染小鼠MEL细胞。用O6-苄基鸟嘌呤(O6-benzylguanine,BG)-卡莫司汀[1,3-bis(2-chloroethyl)-1-nitrosourea,BCNU]联合筛选以富集感染的阳性细胞,用酶联免疫吸附测定方法检测目的基因的表达,评估不同基因调控元件组合对重组慢病毒载体所携带的目的基因表达的影响。结果经限制性内切酶谱分析和序列测定,构建的慢病毒载体结构正确;三质粒系统瞬时转染人肾胚胎293T细胞包装的重组自灭活慢病毒可通过长时间高速离心有效浓缩;用50μmol/L BG-50μmol/L BCNU联合筛选可有效富集感染的阳性细胞;经环六亚甲基双乙酰胺(N,N’-hexamethyle-nebisacetamide,HMBA)诱导第8天的106个小鼠MEL细胞中表达的重组人凝血因子Ⅸ平均质量浓度达到正常水平的3.8%(191 ng/ml)。结论通过适当减少调控元件优化慢病毒载体不仅有助于提高携带的外源基因片段长度、增加载体制备的有效性、实现温和提高目的基因产物达到治疗水平需求量的目的,而且为开展以红细胞特异性表达载体介导的携带蛋白类药物的红细胞治疗以及非血液病基因治疗奠定临床前的研究基础。更多还原

【Abstract】 Objective To construct and optimize recombinant lentiviral vectors(LV) for recombinant gene expression driven by erythroid-specific gene regulatory elements,to investigate their expression capacity in murine erythroleukemia(MEL) cells induced by hexamethylene bisacetamide(HMBA) to terminal erythroid differentiation.Methods The length of erythroid-specific β-globin promoter gene was modified,DNase Ⅰ hypersensitive site(HS) of β-globin gene cluster locus control region was recombined,the 3′-globin enhancer or β-globin intervening sequence IVS2 was deleted.The recombinant gene-human factor Ⅸ(hFⅨ) expression was driven by β-globin in LV vectors with different deletions and recombination of gene regulatory elements.293T cells were co-transfected by transfer,packaging and pseudo-envelope plasmids.The titers of LV vectors were quantified by real-time PCR method.The MEL cells transduced by these LV vectors were enriched by positive selection with various concentrations of O6-benzylguanine(BG) and 1,3-bis(2-chloroethyl)-1-nitrosourea(BCNU).The hFⅨ gene expression level was determined by enzyme-linked immunosorbent assay(ELISA) after BG-BCNU selection and HMBA induction.The contribution of these gene regulatory elements in erythroid-specific gene expression was evaluated.Results The structures of these LV vectors were confirmed by restriction digestion analysis,as well as DNA sequencing.The recombinant LV vectors obtained through tri-plasmids transient transfection of human embryonic kidney 293T cells were effectively concentrated by overnight high-speed centrifugation.The transduced cells were enriched effectively by BG-BCNU selection(50 μmol/L for both dugs).High level of hFⅨ expression was achieved on 8th day after induction by HMBA in 106 MEL cells transduced by LV vectors with various gene regulatory elements,with average concentration of 191 ng/ml,a level equivalent of 3.8% of physiological level in human.Conclusion Optimization of erythroid-specific LV vector through deletion of various gene regulatory elements is critical for increasing gene carrying capacity of the LV vector and achieving better vector production titer.High levels of erythroid-specific recombinant gene expression were achieved in MEL cells transduced with all of the LV vectors with various deletions in gene regulatory regions,indicating that it is possible to develop a more effective erythroid-specific gene expression vector which can satisfy therapeutic requirement and can be produced effectively.Our current work is a very important step for future application of erythroid-specific expression system in developing therapies in the fields of protein drug carriers,vaccines and gene therapies by erythrocytes.更多还原