Urbinati Fabrizia, Arumugam Paritha, Higashimoto Tomoyasu, Perumbeti Anil, Mitts Kyle, Xia Ping, Malik Punam
Division of Experimental Hematology and Cancer Biology, Cincinnati Children's Hospital Medical Center, University of Cincinnati, Cincinnati, Ohio 45229-3039, USA.
Mol Ther. 2009 Sep;17(9):1527-36. doi: 10.1038/mt.2009.89. Epub 2009 Apr 21.
Self-inactivating (SIN) lentiviruses flanked by the 1.2-kb chicken hypersensitive site-4 (cHS4) insulator element provide consistent, improved expression of transgenes, but have significantly lower titers. The mechanism by which this occurs is unknown. Lengthening the lentiviral (LV) vector transgene cassette by an additional 1.2 kb by an internal cassette caused no further reduction in titers. However, when cHS4 sequences or inert DNA spacers of increasing size were placed in the 3'-long terminal repeat (LTR), infectious titers decreased proportional to the length of the insert. The stage of vector life cycle affected by vectors carrying the large cHS4 3'LTR insert was compared to a control vector: there was no increase in read-through transcription with insertion of the 1.2-kb cHS4 in the 3'LTR. Equal amount of full-length viral mRNA was produced in packaging cells and viral assembly/packaging was unaffected, resulting in comparable amounts of intact vector particles produced by either vectors. However, LV vectors carrying cHS4 in the 3'LTR were inefficiently processed following target-cell entry, with reduced reverse transcription and integration efficiency, and hence lower transduction titers. Therefore, vectors with large insertions in the 3'LTR are transcribed and packaged efficiently, but the LTR insert hinders viral-RNA (vRNA) processing and transduction of target cells. These studies have important implications in design of integrating vectors.
两侧带有1.2 kb鸡高敏位点4（cHS4）绝缘子元件的自失活（SIN）慢病毒可使转基因表达持续且得到改善，但滴度显著降低。其发生机制尚不清楚。通过内部盒式结构使慢病毒（LV）载体转基因盒式结构再延长1.2 kb，滴度未进一步降低。然而，当将cHS4序列或长度不断增加的惰性DNA间隔区置于3'长末端重复序列（LTR）中时，感染滴度随插入片段长度成比例下降。将携带大cHS4 3'LTR插入片段的载体对载体生命周期阶段的影响与对照载体进行比较：在3'LTR中插入1.2 kb cHS4后通读转录没有增加。包装细胞中产生等量的全长病毒mRNA，病毒组装/包装不受影响，两种载体产生的完整载体颗粒数量相当。然而，3'LTR中携带cHS4的LV载体在进入靶细胞后加工效率低下，逆转录和整合效率降低，因此转导滴度较低。因此，3'LTR中有大插入片段的载体转录和包装效率高，但LTR插入片段会阻碍病毒RNA（vRNA）加工和靶细胞转导。这些研究对整合载体的设计具有重要意义。
