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，我们制定了一种新颖的策略来选择性扩展转导的造血细胞。编码G-CSF受体（G-CSFR）和激素结合结构域（ER）之间编码嵌合受体（Deltagcrer）的融合基因是“选择性放大器基因（SAG）”的原型。 G-CSFR被用作信号发生器，并删除了G-CSF结合域，以不对G-CSF.ER-HBD响应，用作分子开关来控制雌激素（E_2）依赖性方式中融合蛋白的活性。尽管当BAIF3细胞通过该下垂转导BAIF3细胞时，可以实现E_2诱导的生长，但在B2处理后，它也诱导了转导的32D细胞的分化。由于仅需要一个生长信号，因此我们修改了Deltagcrer基因以减轻分化信号。 PHE在嵌合受体中被取代为Tyr7O3。当所得的SAG（Deltay7O3F-Grer基因）在32d中表达时，在E_2处理后观察到持续的生长，而持续的生长则最小。研究结果表明，deltay7o3f-gcrbr基因可以用作更理想的sag。在加法中，突变体ER（TMR）专门与合成配体4-羟基氧法（TM）专门结合，用作另一个分子开关。当在BA/F3细胞中表达GCRTMR时，观察到TM依赖性生长，对E_2的反应很少。当将SAG方法应用于转导的造血细胞的体内扩增时，GCRTMR/TM系统可能很有价值，因为可以消除内源性E_2的影响。