Detailed comparison of retroviral vectors and promoter configurations for stable and high transgene expression in human induced pluripotent stem cells
Detailed comparison of retroviral vectors and promoter configurations for stable and high transgene expression in human induced pluripotent stem cells
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DOI：
10.1038/gt.2017.20
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发表时间：
2017-05-01
期刊：
影响因子：
5.1
通讯作者：
Schambach, A.
中科院分区：
文献类型：
--
作者：
Hoffmann, D.;Schott, J. W.;Schambach, A.
通过逆转录病毒载体的基因传递纠正患者特异性诱导多能干细胞(IPSC)，为单基因疾病的治疗提供了新的前景。基因修饰的IPSC克隆可以被筛选出安全的整合位点，并分化为感兴趣的可移植细胞。然而，目前的瓶颈是表观遗传载体沉默。为了确定最适合IPSC的逆转录病毒表达系统，我们系统地比较了来自不同逆转录病毒属的载体、不同启动子及其与普遍存在的染色质开放元件(UCOE)的结合，以及几种包膜伪型。以水泡性口炎病毒糖蛋白为假型的慢病毒载体(LV)优于伽玛逆转录病毒载体和甲型逆转录病毒载体及其他包膜。延长因子1α短链(EFS)启动子介导了最强健的表达，而有效但更容易沉默的脾病灶形成病毒(SFFV)启动子的表达水平较低。两个生理启动子和一个病毒启动子并列的全长(A2UCOE)和最小UCOE(CBX3)UCOE以同样的效率减少了转基因沉默。然而，启动子特异性表达水平的下降并没有完全被阻止。转基因阳性的IPSC分化为内皮细胞后，A2UCOE。EFS和CBX3。与这里测试的所有其他载体相比，EFS载体在更大比例的细胞中保持了最高的转基因表达。UCOE的功能减弱了，但没有完全抵消，媒介沉默和改进的可能性仍然存在。然而，CBX3。LV背景下的EFS对人IPSC及其后代的高滴度生产和长期遗传修饰都显示出最有希望的启动子和载体构型。
Correction of patient-specific induced pluripotent stem cells (iPSC) upon gene delivery through retroviral vectors offers new treatment perspectives for monogenetic diseases. Gene-modified iPSC clones can be screened for safe integration sites and differentiated into transplantable cells of interest. However, the current bottleneck is epigenetic vector silencing. In order to identify the most suitable retroviral expression system in iPSC, we systematically compared vectors from different retroviral genera, different promoters and their combination with ubiquitous chromatin opening elements (UCOE), and several envelope pseudotypes. Lentiviral vectors (LV) pseudotyped with vesicular stomatitis virus glycoprotein were superior to gammaretroviral and alpharetroviral vectors and other envelopes tested. The elongation factor 1 alpha short (EFS) promoter mediated the most robust expression, whereas expression levels were lower from the potent but more silencing-prone spleen focus forming virus (SFFV) promoter. Both full-length (A2UCOE) and minimal (CBX3) UCOE juxtaposed to two physiological and one viral promoter reduced transgene silencing with equal efficiency. However, a promoter-specific decline in expression levels was not entirely prevented. Upon differentiation of transgene-positive iPSC into endothelial cells, A2UCOE. EFS and CBX3. EFS vectors maintained highest transgene expression in a larger fraction of cells as compared with all other constructs tested here. The function of UCOE diminished, but did not fully counteract, vector silencing and possibilities for improvements remain. Nevertheless, the CBX3. EFS in a LV background exhibited the most promising promoter and vector configuration for both high titer production and long-term genetic modification of human iPSC and their progeny.