建立携带左旋天门冬酰胺酶的红细胞特异性表达系统

目的:建立基因修饰红细胞输送左旋天门冬酰胺酶(L-ASNase)的体系。方法:采用2A联体技术将L-ASNase和红色单体荧光蛋白基因分别置于红细胞特异性基因调控元件或非组织特异性短延长因子1α启动子控制下,构建慢病毒载体,分别包装成组织特异性重组慢病毒或非组织特异性重组慢病毒,测定病毒滴度,并分别感染靶细胞,用六氧苄基鸟嘌呤/卡莫司汀联合筛选以富集感染的小鼠红白血病阳性细胞;用六亚甲基二乙酰胺诱导富集的阳性细胞向红细胞分化,流式细胞术检测红色单体荧光蛋白报告基因的表达,荧光显微术观察基因表达产物在细胞中的定位,免疫印迹分析L-ASNase的表达水平,评估在红细胞分化过程中组织特异性重组慢病毒的表达优势。结果:经限制性内切酶谱分析和序列测定,构建的重组慢病毒载体结构正确;免疫荧光结果显示在非组织特异性慢病毒感染的HeLa细胞中,L-ASNase主要表达在细胞质,单体红色荧光蛋白主要表达在细胞核内,提示2A序列通过自裂解使同一读框中的2个基因获得了表达;用50μmol/L六氧苄基鸟嘌呤-50μmol/L卡莫司汀联合筛选,可有效富集感染的小鼠红白血病阳性细胞;经六亚甲基二乙酰胺诱导,第7 d的MEL细胞中表达的重组L-ASNase浓度达0.3 U/mg总蛋白。结论:红细胞特异性慢病毒载体可以在小鼠红白血病细胞向红细胞分化过程中使携带基因的表达逐步增高,优于非组织特异性慢病毒载体。本研究为基因修饰造血干细胞、并通过体外细胞分化的方法大量产生携带L-ASNase的红细胞治疗恶性血液病奠定了临床前的实验基础。

Establishment of Erythroid Expression System Carrying L-Asparagi- nase Gene

／ Objective： To construct the self-inactivating lentiviral vector for recombinant L-asparaginase （L-／ ASNase） gene driven by regulatory elements, to investigate in ~murine erythroleukemia（MEL） cells for their L-／ ASNase-carrying capability in their terminally differentiated stage. Methods： The target genes driven by erthroid-／ specific expression regulatory elements or short elongation factor 1-alpha promoter were expressed from a single reading frame by linking L-ASNase and mCherry with a 2A self-processing peptide derived from the Thosea asigna virus. The 293T cells were co-transfected with the recombinant lentiviral vector together with packaging and pseudo-envelope plasmids. The virus titers were quantified by real-time PCR method. The target cells were infected with recombinant lentivirus, respectively. The transduced MEL cells were enriched effectively by O^6- benzylguanine （BG）/1,3- bis （2- chloroethyl） - 1- nitrosourea （BCNU） selection （50 μmol/L for both drugs）. Flow cytometry was used to detect the expression content of mCherry in infectious cells. The cellular localization of expression produets was observed under fluorescence microscopy. The L-ASNase gene expression levels were determined by Western blot after BG/BCNU selection and hexamethylene bisaeetamide （HMBA） induction. The contribution of the gene regulatory elements in erythroid-specific or non-tissue specific gene expression was evaluated. Results： The structures of these lentiviral vector vectors were confirmed by restriction digestion analysis, as well as DNA sequencing. The titer of virus was effectively increased by overnight high-speed centrifugation. LASNase was localized principally in cytoplasm and mCherry was expressed mainly in nuclear in non-tissue specific lentivirus infectious HeLa cells, thus the 2A sequence efficiently facilitates L-ASNase and mCherry expression from a single ORF. The infectious MEL cells were enriched effectively by BG/BCNU selection. The concentration of L-ASNase expression was achieved 0.3 U/mg in total protein on 7th day after induction by HMBA in MEL ceils infected by virus with erythroid-specific gene regulatory elements. Conclusion： The gradually increase levels of erythroid-restricted transgene expression were achieved during inducer-mediated erythroid differentiation in MEL ceils infected with the erythroid-specific recombinant lentivirus, the expression trend was better than non-tissue specific lentivirus infectious. Our current work is a very important step for future mass production L-ASNase-carrying erythrocytes through hematopoietic stem cell transduction and in vitro erythroid differentiation and to study for their anti-cancer effect.