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Development of GFP-tagged retrovirus vectors for gene therapy

用于基因治疗的 GFP 标记逆转录病毒载体的开发

基本信息

批准号：
09670829
负责人：
KUME Akihiro
金额：
$ 1.92万
依托单位：
Jichi Medical School
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1997
资助国家：
日本
起止时间：
1997 至 1998
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-09670829/
关键词：
gene therapy retrovirus vector hematopoietic stem cell green fluorescent protein green flnogescent protein

项目摘要

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.

为了开发和改进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标中，并定量跟踪体内转基因表达。为此，我们构建了一种逆转录病毒载体，该载体能够用绿色荧光蛋白（GFP）标记细胞，同时引入治疗基因。在本研究中，我们构建了一种双顺反子逆转录病毒载体，其中包含人CD24基因作为第一个顺反子和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GFP (EGFP) 基因作为第二个顺反子 (MSCV/CD24-IRES-EGFP)。当我们用MSCV/CD24-IRES-EGFP转导Ba/F3小鼠前B细胞系时，CD24和EGFP的表达在没有选择的情况下稳定维持了6个月以上。该载体还成功转导原代小鼠骨髓细胞。在离体操作后不久的骨髓细胞以及源自感染骨髓的红细胞和粒细胞集落中证实了 CD24/EGFP 表达。最重要的是，MSCV/CD24-IRF、S-EGEP 转导了长期重建的小鼠造血干细胞。将MSCV/CD24-IRES-EGFP转导的骨髓细胞移植到经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，在二次受者中也稳定表达 6 个月。对主要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，所有检查谱系（B 和 T 淋巴、红系、粒细胞和单核细胞）均具有正常的造血分化。总之，MSCV/CD24-JRES-EGFP载体成功转导了能够自我更新和多谱系分化的小鼠造血干细胞。