Stable Transgene Expression in Primitive Human CD34+ Hematopoietic Stem/Progenitor Cells, Using the Sleeping Beauty Transposon System
Stable Transgene Expression in Primitive Human CD34+ Hematopoietic Stem/Progenitor Cells, Using the Sleeping Beauty Transposon System
复制标题
DOI：
10.1089/hum.2009.109
复制
发表时间：
2009-12-01
影响因子：
4.2
通讯作者：
Kohn, Donald B.
中科院分区：
文献类型：
--
作者：
Sumiyoshi, Teiko;Holt, Nathalia G.;Kohn, Donald B.
睡美人（SB）转座子介导的整合已被证明可以在广泛的宿主细胞中实现长期的转基因表达。在本研究中，我们通过两种方法改进了SB转座子介导的转导人CD 34（+）干/祖细胞的基因转移系统：（1）为了提高转座效率，使用SB的高活性突变体HSB;（2）为了提高SB转座酶和转座子携带的转基因盒的表达，评估了不同的病毒和细胞启动子。SB组分通过核穿孔反式递送到靶细胞中。SB转座子介导的整合功效通过整合的转基因（增强型绿色荧光蛋白[eGFP]）在体外和体内的表达来评估。在纯化的人脐带血CD 34（+）细胞中，HSB在比原始SB转座酶多近7倍的细胞中实现了长期转基因表达。在Jurkat人T细胞中使用人延伸因子1 α（EF 1-α）启动子实现的eGFP表达水平（5倍）显著高于使用修饰的骨髓增生肉瘤病毒长末端重复序列增强子启动子（MNDU 3）实现的水平;相反，MNDU 3启动子在K-562髓样细胞中以最高水平表达eGFP。在指导骨髓分化的条件下研究的人CD 34（+）脐带血细胞中，使用EF 1-α启动子表达SB转座酶，结合MNDU 3启动子表达eGFP报告基因，实现了最高的转基因整合和表达。在改进的基因转移到CD 34（+）细胞后，在培养4周以上的时间内，稳定的转基因表达水平达到27%（平均17%; n = 4）。评价SB修饰的人CD 34（+）细胞的移植和分化的体内研究表明，SB修饰的人CD 34（+）细胞在NOD/SCID/γ链（null）（NSG）小鼠中移植并分化成具有eGFP表达的多谱系细胞类型。更重要的是，二次移植研究表明，整合的转基因在更原始的CD 34（+）造血干细胞（HSC）中稳定表达，具有长期的再增殖能力。这项研究表明，改进的HSB基因转移系统可以稳定地将基因整合到原始人HSC中，同时保持干细胞的多能性，这为进一步发展使用造血干细胞的非病毒基因治疗显示了希望。
Sleeping Beauty (SB) transposon-mediated integration has been shown to achieve long-term transgene expression in a wide range of host cells. In this study, we improved the SB transposon-mediated gene transfer system for transduction of human CD34(+) stem/progenitor cells by two approaches: (1) to increase the transposition efficacy, a hyperactive mutant of SB, HSB, was used; (2) to improve the expression of the SB transposase and the transgene cassette carried by the transposon, different viral and cellular promoters were evaluated. SB components were delivered in trans into the target cells by Nucleoporation. The SB transposon-mediated integration efficacy was assessed by integrated transgene (enhanced green fluorescent protein [eGFP]) expression both in vitro and in vivo. In purified human cord blood CD34(+) cells, HSB achieved long-term transgene expression in nearly 7-fold more cells than the original SB transposase. Significantly brighter levels of eGFP expression (5-fold) were achieved with the human elongation factor 1 alpha (EF1-alpha) promoter in Jurkat human T cells, compared with that achieved with the modified myeloproliferative sarcoma virus long terminal repeat enhancer-promoter (MNDU3); in contrast, the MNDU3 promoter expressed eGFP at the highest level in K-562 myeloid cells. In human CD34(+) cord blood cells studied under conditions directing myeloid differentiation, the highest transgene integration and expression were achieved using the EF1-alpha promoter to express the SB transposase combined with the MNDU3 promoter to express the eGFP reporter. Stable transgene expression was achieved at levels up to 27% for more than 4 weeks of culture after improved gene transfer to CD34(+) cells (average, 17%; n = 4). In vivo studies evaluating engraftment and differentiation of the SB-modified human CD34(+) cells demonstrated that SB-modified human CD34(+) cells engrafted in NOD/SCID/gamma chain(null) (NSG) mice and differentiated into multilineage cell types with eGFP expression. More importantly, secondary transplantation studies demonstrated that the integrated transgene was stably expressed in more primitive CD34(+) hematopoietic stem cells (HSCs) with long-term repopulating capability. This study demonstrates that an improved HSB gene transfer system can stably integrate genes into primitive human HSCs while maintaining the pluripotency of the stem cells, which shows promise for further advancement of non-virus-based gene therapy using hematopoietic stem cells.