Retrovirus-mediated gene transfer of hematopoietic stimulating factor receptor cDNA into MDS cells and gene therapy

逆转录病毒介导的造血刺激因子受体cDNA基因转移至MDS细胞及基因治疗

• 批准号: 12670980
• 负责人: OHNISHI Kazunori
• 金额: $ 2.18万
• 依托单位: Hamamatsu University School of Medicine
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2000
• 资助国家: 日本
• 起止时间: 2000 至 2001
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-12670980/
• 关键词: myelodysplastic syndrome; gene therapy; granulocyte colony-stimulating factor receptor; retroviral vector; granulocytic differentiation; SH-PTP1; 遺伝子導入; チロシンリン酸化酵素; エリスロポエチン受容体遺伝子; トロンポボエチン受容体遺伝子

Myelodysplastic syndrome (MDS) are clonal disorders in which the proper differentiation of hematopoietic stem cells is impaired. There is no effective treatment for this stem cell disorder at present. In an attempt to find a new strategy that promotes the differentiation of MDS blast cells, we tried retroviral transduction of granulocyte colony-stimulating factor receptor (G-CSFR) into an interleukin-3-dependent MDS cell line, MDS-L. Ectopic expression of human G-CSFR cDNA in MDS-L cells gave rise to granulocytic differentiation by G-CSF stimulation. G-CSF caused the transformants expressing G-CSFR to display a morphological characteristics of mature granulocytes. These results demonstrate that MDS-L cells ectopically expressing G-CSFR are induced to granulocytic differentiation upon exposure to G-CSF.Next, we showed that SH-PTP1 bound selectively to phosphorylated tyrosine at 729 ammo1"acid position (pY729) in the cytoplasmic domain of G-CSFR through its SH2 domain. This interaction mediated the dephosphorylation and inactivation of JAK2. Recruitment of SH-PTP1 to G-CSFR containing pY729, induced by G-CSF binding, caused dephosphorylation of JAK2. Compared to wild-type G-CSFR transfectants, stable transfectants of a mutant G-CSFR lacking Y729 allowed proliferation of cells at the one-fourth concentration of G-CSF and remarkably prolonged hyperphosphorylation of both JAK2 and G-CSFR. We conclude that SH-PTP1 play an important role in down-modulation of proliferation signals generated by the activated G-CSFR/JAK2 complexes.Our experimental design provides new insight into the strategy of gene therapy for hamatologic disorders by retrovirus-mediated gene transfer.

骨髓增生异常综合征（MDS）是造血干细胞的正常分化受损的克隆性疾病。目前还没有有效的治疗这种干细胞疾病的方法。为了寻找促进骨髓增生异常综合症原始细胞分化的新策略，我们尝试将粒细胞集落刺激因子受体（G-CSFR）逆转录病毒转导到白细胞介素-3依赖性骨髓增生异常综合症细胞系MDS-L中。人G-CSFR cDNA在MDS-L细胞中的异位表达通过G-CSF刺激引起粒细胞分化。G-CSF使表达G-CSFR的转化子呈现成熟粒细胞的形态特征。这些结果表明，异位表达G-CSFR的MDS-L细胞在暴露于G-CSF时被诱导向粒细胞分化。接下来，我们表明SH-PTP 1通过其SH 2结构域选择性地结合G-CSFR胞质结构域中729氨基酸位置（pY 729）的磷酸化酪氨酸。这种相互作用介导JAK 2的去磷酸化和失活。G-CSF结合诱导SH-PTP 1向含有pY 729的G-CSFR的募集，引起JAK 2的去磷酸化。与野生型G-CSFR转染子相比，缺乏Y 729的突变型G-CSFR的稳定转染子允许细胞在四分之一浓度的G-CSF下增殖，并且显著延长JAK 2和G-CSFR的过度磷酸化。结论SH-PTP 1在下调G-CSFR/JAK 2复合物的增殖信号中起重要作用，为逆转录病毒介导的基因转移治疗血液病提供了新的思路。

项目成果

Satoki Nakamura, Kazunori Ohnishi, Hotoshi Yoshida, Kaori Shinjo, Akihiro Takeshita, Ryuzo Ohno, Kaoru Tohyama, Yukio Koide: "Retrovirus-mediated gene transfer of granuloc yte-colony stimulating factor receptor (G-CSFR) cDNA into MDS cells and induction o

Satoki Nakamura、Kazunori Ohnishi、Hotoshi Yoshida、Kaori Shinjo、Akihiro Takeshita、Ryuzo Ohno、Kaoru Tohyama、Yukio Koide：“逆转录病毒介导的粒细胞集落刺激因子受体 (G-CSFR) cDNA 基因转移至 MDS 细胞并诱导

Satoki Nakamura: "Retrovirus-mediated gene transfer of granulocyte-colony stimulating factor receptor (G-CSFR) cDNA into MDS cells and induction of their differentiation by G-CSF"Cytokines, Cellular, and Molecular Therapy. 6. 61-70 (2000)

Satoki Nakamura：“逆转录病毒介导的粒细胞集落刺激因子受体 (G-CSFR) cDNA 基因转移至 MDS 细胞，并通过 G-CSF 诱导其分化”细胞因子、细胞和分子治疗。

Satoki Nakamura: "Retrovirus-mediated gene transfer of granulocyte-colony stimulating factor receptor (G-CSFR) cDNA into MDS cells and induction of their differentiation by G-CSF."Cytokines,Cellular,and Molecular Therapy. 6. 61-70 (2000)

Satoki Nakamura：“逆转录病毒介导的粒细胞集落刺激因子受体 (G-CSFR) cDNA 基因转移至 MDS 细胞，并通过 G-CSF 诱导其分化。”细胞因子、细胞和分子治疗。