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Development of a novel regulatory gene for in vivo & in vitro expansion of transduced hematopoietic stem cellss

开发一种新型体内调节基因

基本信息

批准号：
09557087
负责人：
OZAWA Keiya
金额：
$ 5.57万
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 (B)
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-09557087/
关键词：
Gene therapy Gene transfer Hematopoietic stem cell Selective amplifier gene G-CSF receptor Estrogen receptor in vivo expansion ex vivo expansion 対外増幅

项目摘要

To overcome the low efficiency of gene transfer into hematopoietic stem cells, we have developed a novel strategy for selective expansion of transduced hematopoietic cells. A fusion gene encoding a chimeric receptor (DELTAGCRER) between G-CSF receptor (G-CSFR) and hormone-binding domain (HBD) of estrogen receptor (ER) was constructed as a prototype of "selective amplifier genes (SAG)". G-CSFR was employed as a signal generator and the G-CSF-binding domain was deleted not to respond to G-CSF.ER-HBD was employed as a molecular switch to control the activity of fusion protein in estrogen (E_2)-dependent manner. Although E_2-inducible growth was achieved when BaIF3 cells were transduced with this SAG, it also induced differentiation in transduced 32D cells upon B2 treatment. Since only a growth signal is required, we modified the DELTAGCRER gene to attenuate the differentiation signal. Phe was substituted for Tyr7O3 in the chimeric receptor. When the resultant SAG (DELTAY7O3F-GCRER gene) was expressed in 32D, sustained growth was observed with minimal differentiation upon E_2 treatment. The findings suggest that DELTAY7O3F-GCRBR gene can function as a more desirable SAG.In addition, a mutant ER (TmR), which specifically binds to a synthetic ligand 4-hydroxytamoxifen (Tm), was used as another molecular switch. When GCRTmR was expressed in Ba/F3 cells, Tm-dependent growth was observed with little response to E_2. The GCRTmR/Tm system may be valuable when the SAG method is applied to in vivo amplification of transduced hematopoietic cells, because the influences of endogenous E_2 can be eliminated.

为了克服基因转移到造血干细胞中的低效率，我们已经开发了一种新的策略，用于选择性扩增转导的造血细胞。以G-CSF受体（G-CSFR）与雌激素受体（ER）的结合域（HBD）的嵌合受体（DELTAGCRER）为原型构建了选择性放大基因（SAG）。以G-CSFR为信号发生器，缺失G-CSF结合区，使其对G-CSF无反应，以ER-HBD为分子开关，以雌激素（E_2）依赖方式调控融合蛋白的活性。用此SAG转导BaIF 3细胞，虽然能达到E_2诱导的生长，但在B_2处理时，它也能诱导转导的32 D细胞分化。由于只需要生长信号，我们修饰了DELTAGCRER基因以减弱分化信号。Phe取代嵌合受体中的Tyr 7 O3。当所得到的SAG（DELTAY 7 O3 F-GCRER基因）在32 D中表达时，观察到持续的生长，并且在E_2处理时分化最小。另外，一个能与合成配体4-羟基他莫昔芬（Tm）特异性结合的突变型ER（TmR）被用作另一个分子开关。当GCRTmR在Ba/F3细胞中表达时，观察到Tm依赖性生长，对E_2几乎没有反应。GCRTmR/Tm系统可消除内源性E_2的影响，在SAG法应用于转基因造血细胞的体内扩增中具有一定的应用价值。