Division of Research Immunology/Bone Marrow Transplantation, Saban Research Institute of Childrens Hospital Los Angeles, Los Angeles, CA 90027, USA.
Hum Gene Ther. 2009 Dec;20(12):1607-26. doi: 10.1089/hum.2009.109.
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 1alpha (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.
睡眠美人 (SB) 转座子介导的整合已被证明可在广泛的宿主细胞中实现长期转基因表达。在这项研究中，我们通过两种方法改进了 SB 转座子介导的基因转移系统，以转导人 CD34(+) 干细胞/祖细胞：(1) 为了提高转座效率，使用了超活突变体 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。通过核转染将 SB 组件递送至靶细胞中。通过体外和体内整合转基因 (增强型绿色荧光蛋白 [eGFP]) 表达评估 SB 转座子介导的整合效率。在纯化的人脐血 CD34(+) 细胞中，HSB 使近 7 倍的细胞长期表达转基因。与经修饰的髓性白血病肉瘤病毒长末端重复增强子-启动子 (MNDU3) 相比，在 Jurkat 人 T 细胞中，使用人延伸因子 1alpha (EF1-alpha) 启动子可实现 eGFP 表达的显著增强 (5 倍)；相反，在 K-562 髓样细胞中，MNDU3 启动子表达 eGFP 的水平最高。在导向髓样分化的条件下研究的人 CD34(+) 脐血细胞中，使用 EF1-alpha 启动子表达 SB 转座酶并使用 MNDU3 启动子表达 eGFP 报告基因，可实现最高的转基因整合和表达。经过改良的基因转移到 CD34(+) 细胞后，稳定的转基因表达水平高达 27%，培养 4 周以上(平均 17%；n = 4)。评估 SB 修饰的人 CD34(+) 细胞植入和分化的体内研究表明，SB 修饰的人 CD34(+) 细胞植入 NOD/SCID/gamma 链缺失 (NSG) 小鼠中，并分化为具有 eGFP 表达的多谱系细胞类型。更重要的是，二次移植研究表明，整合的转基因在具有长期重编程能力的更原始的 CD34(+) 造血干细胞 (HSCs) 中稳定表达。这项研究表明，改良的 HSB 基因转移系统可以稳定地将基因整合到原始的人类 HSCs 中，同时保持干细胞的多能性，这为进一步推进基于造血干细胞的非病毒基因治疗提供了希望。
