Development of GFP-tagged retrovirus vectors for gene therapy

为了开发和改进靶向造血细胞的基因治疗载体和基因转移方案，必须准确地评估基因转移到靶点如造血干细胞中并定量地跟踪体内转基因表达。为此，我们构建了一个以人CD 24基因为第一顺反子，以小核糖核酸病毒内部核糖体进入位点（IRES）控制的增强型绿色荧光蛋白（EGFP）基因为第二顺反子的双顺反子逆转录病毒载体（MSCV/CD 24-IRES-EGFP）。当我们用MSCV/CD 24-IRES-EGFP转导Ba/F3小鼠前B细胞系时，CD 24和EGFP的表达稳定持续超过6个月而不进行选择。该载体还成功地转导了原代小鼠骨髓细胞。在离体操作后立即在骨髓细胞中证实了CD 24/EGFP表达，以及来自感染骨髓的红细胞和粒细胞集落。MSCV/CD 24-IRF、S-EGEP转导的小鼠造血干细胞具有长期增殖能力。将MSCV/CD 24-IRES-EGFP转导的骨髓细胞移植到致死辐射的受体小鼠中，并监测转基因表达。CD 24和EGEP在外周血中稳定表达6个月，在第二受体中稳定表达6个月。对初次接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，所有检查的谱系（B-和T-淋巴、红细胞、粒细胞和单核细胞）的造血分化正常。MSCV/CD 24-JRES-EGFP载体成功转染了具有自我更新和多向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.