Development of GFP-tagged retrovirus vectors for gene therapy

为了开发和完善针对造血细胞的基因治疗载体和基因转移方案，必须准确评估基因转移到造血干细胞等靶点，并定量跟踪转基因在体内的表达。为此，我们构建了一种逆转录病毒载体，可以同时用绿色荧光蛋白（GFP）标记细胞并引入治疗基因。本研究构建了以人CD24基因为第一顺子，以小核糖核酸病毒衍生的内部核糖体进入位点（IRES）控制的增强型绿色荧光蛋白（EGFP）基因为第二顺子的双顺子逆转录病毒载体（MSCV/CD24-IRES-EGFP）。用MSCV/CD24- ires -EGFP转导Ba/F3小鼠b前细胞系，CD24和EGFP的表达稳定持续6个月以上。该载体也成功地转导了原代小鼠骨髓细胞。CD24/EGFP随即在骨髓细胞中表达。离体操作，以及从受感染骨髓中提取的红细胞和粒细胞菌落。最重要的是，MSCV/CD24-IRF，S-EGEP转导了长期再生的小鼠造血干细胞。将MSCV/ cd24 - ires - egfp转导的骨髓细胞移植到致死照射的受体小鼠中，并监测转基因表达。CD24和EGEP在外周血中稳定表达6个月，在继发性受体中稳定表达6个月。对初次受者的淋巴造血组织（骨髓、外周血、胸腺和脾脏）的详细分析显示，所有被检查谱系（B淋巴和t淋巴、红细胞、粒细胞和单核细胞）的造血分化正常。综上所述，MSCV/CD24-JRES-EGFP载体成功转染了具有自我更新和多系分化能力的小鼠造血干细胞。

To develop and improve gene therapy vectos and gene transfer protocols targeting hematopoietic cells, it is essential to accurately assess gene transfer into the targets such as hematopoietic stem cells and to quantitatively track transgene expression in vivo. For this aim, we constructed a retrovirus vector which enables cell marking with green fluorescent protein (GFP) and introducing a therapeutic gene simultaneously.In the present study, we constructed a bicistronic retrovirus vector containing the human CD24 gene as the first cistron and the picornavirus-derived internal ribosome entry site (IRES)-controlled the enhanced GFP (EGFP) gene as the second cistron (MSCV/CD24-IRES-EGFP). When we transduced Ba/F3 murine pre-B cell line with MSCV/CD24-IRES-EGFP, expression of CD24 and EGFP was stably sustained for more than 6 months without selection. This vector also transduced primary murine bone marrow cells successfully. CD24/EGFP expression was confirmed in the bone marrow cells just after .ex vivo manipulation, as well as erythroid and granulocyte colonies derived from the infected bone marrow. Above all, MSCV/CD24-IRF,S-EGEP transduced long-term repopulating murine hematopoletic stem cells. MSCV/CD24-IRES-EGFP-transduced bone marrow cells were transplanted into lethally irradiated recipient mice, and transgene expression was monitored. Stable expression of CD24 and EGEP was demonstrated in the peripheral blood for 6 months, and in the secondary recipients for another 6 months. Detailed analysis of the lymphohematopoietic tissues (bone marrow, peripheral blood, thymus and spleen) in the primary recipients revealed normal hematopoietic differentiation in all the examined lineages (B- and T-lymphoid, erythroid, granulocytic and monocytic). Taken together, MSCV/CD24-JRES-EGFP vector successfully transduded tine murine heinatopoietic stem cells capable of self-renewal and multilineage differentiation.