Schambach Axel, Galla Melanie, Maetzig Tobias, Loew Rainer, Baum Christopher
1Department of Experimental Hematology, Hannover Medical School, Hannover, Germany.
Mol Ther. 2007 Jun;15(6):1167-73. doi: 10.1038/sj.mt.6300152. Epub 2007 Apr 3.
Adverse events relating to insertional mutagenesis have reinforced the interest in self-inactivating (SIN) gamma-retroviral and lentiviral vectors without enhancer-promoter sequences in the U3 region of the long terminal repeats. However, SIN vectors suffer from leaky transcriptional termination, increasing the probability of read-through into cellular genes. To improve 3' end processing, we incorporated seven upstream polyadenylation enhancer elements (or upstream sequence elements, USEs) derived from viral or cellular genes into the 3' U3 region of gamma-retroviral and lentiviral SIN vectors. A 100-base-pair sequence representing a recombinant direct repeat of the USE derived from simian virus 40 (2xSV USE) gave the best results, improving both titer and gene expression. In both gamma-retroviral and lentiviral SIN vectors, the 2xSV USE partially substituted for effects provided by the much larger post-transcriptional regulatory element derived from woodchuck hepatitis virus (wPRE). By northern blot and reporter assays, we found that the 2xSV USE greatly improved proper messenger RNA (mRNA) processing at the retroviral termination signal. Importantly, the 2xSV USE was superior to the wPRE in suppressing transcriptional read-through, improving not only vector efficiency but potentially also biosafety.
与插入诱变相关的不良事件，增强了人们对长末端重复序列U3区域不含增强子-启动子序列的自失活（SIN）γ逆转录病毒载体和慢病毒载体的兴趣。然而，SIN载体存在转录终止渗漏问题，增加了通读进入细胞基因的可能性。为了改善3'端加工，我们将七个源自病毒或细胞基因的上游聚腺苷酸化增强元件（或上游序列元件，USEs）整合到γ逆转录病毒和慢病毒SIN载体的3' U3区域。一个代表源自猿猴病毒40的USE重组直接重复序列的100个碱基对序列（2xSV USE）产生了最佳结果，提高了滴度和基因表达。在γ逆转录病毒和慢病毒SIN载体中，2xSV USE部分替代了源自土拨鼠肝炎病毒（wPRE）的大得多的转录后调控元件所提供的效应。通过Northern印迹和报告基因检测，我们发现2xSV USE极大地改善了逆转录病毒终止信号处信使RNA（mRNA）的正确加工。重要的是，2xSV USE在抑制转录通读方面优于wPRE，不仅提高了载体效率，还可能提高了生物安全性。
