Manic Gwenola, Maurin-Marlin Aurélie, Galluzzi Lorenzo, Subra Frédéric, Mouscadet Jean-François, Bury-Moné Stéphanie
Laboratoire de Biologie et de Pharmacologie Appliquée, UMR 8113 CNRS, Ecole Normale Supérieure de Cachan, FR-94230 Cachan, France.
Hum Gene Ther Methods. 2012 Apr;23(2):84-97. doi: 10.1089/hgtb.2011.154. Epub 2012 Mar 28.
Gene transfer for research or gene therapy requires the design of vectors that allow for adequate and safe transgene expression. Current methods to modulate the safety and expression profile of retroviral vectors can involve the insertion of insulators or scaffold/matrix-attachment regions in self-inactivating long terminal repeats (SIN-LTRs). Here, we generated a set of lentiviral vectors (with internal CMV or PGK promoter) in which we inserted (at the level of SIN-LTRs) sequences of avian (i.e., chicken hypersensitive site-4, cHS4), human (i.e., putative insulator and desert sequence), or bacterial origin. We characterized them with respect to viral titer, integration, transduction efficiency and transgene expression levels, in both integrase-proficient and -deficient contexts. We found that the cHS4 insulator enhanced transgene expression by a factor of 1.5 only when cloned in the antisense orientation. On the other hand, cHS4 in the sense orientation as well as all other inserts decreased transgene expression. This attenuation phenomenon persisted over long periods of time and did not correspond to extinction or variegation. Decreased transgene expression was associated with lower mRNA levels, yet RNA stability was not affected. Insertions within the SIN-LTRs may negatively affect transgene transcription in a direct fashion through topological rearrangements. The lentiviral vectors that we generated constitute valuable genetic tools for manipulating the level of transgene expression. Moreover, this study demonstrates that SIN-LTR inserts can decrease transgene expression, a phenomenon that might be overcome by modifying insert orientation, thereby highlighting the importance of careful vector design for gene therapy.
用于研究或基因治疗的基因转移需要设计能够实现充足且安全的转基因表达的载体。目前调节逆转录病毒载体安全性和表达谱的方法可能涉及在自失活长末端重复序列（SIN-LTRs）中插入绝缘子或支架/基质附着区域。在此，我们构建了一组慢病毒载体（带有内部CMV或PGK启动子），并在其中（在SIN-LTRs水平）插入了禽类（即鸡高敏位点4，cHS4）、人类（即假定的绝缘子和荒漠序列）或细菌来源的序列。我们在整合酶功能正常和缺陷的情况下，对它们的病毒滴度、整合、转导效率和转基因表达水平进行了表征。我们发现，只有当以反义方向克隆时，cHS4绝缘子才能将转基因表达提高1.5倍。另一方面，正义方向的cHS4以及所有其他插入片段均降低了转基因表达。这种衰减现象长期存在，且与基因沉默或斑驳现象无关。转基因表达降低与较低的mRNA水平相关，但RNA稳定性未受影响。SIN-LTRs内的插入可能通过拓扑重排直接对转基因转录产生负面影响。我们构建的慢病毒载体是用于调控转基因表达水平的有价值的遗传工具。此外，本研究表明SIN-LTR插入片段可降低转基因表达，这一现象可能通过改变插入方向来克服，从而突出了精心设计载体对于基因治疗的重要性。
