Poluri Ananthalakshmi, Sutton Richard E
Department of Molecular Virology & Microbiology, Baylor College of Medicine, Houston, Texas 77030, USA.
Mol Ther. 2008 Feb;16(2):378-86. doi: 10.1038/sj.mt.6300370. Epub 2007 Dec 11.
Gene transfer vectors encoding short hairpin RNAs (shRNAs) are useful in deciphering gene function, and are being considered for therapeutic knockdown of target genes in humans. We constructed HIV-based vectors encoding shRNA against HIV coreceptor chemokine (C-C motif) receptor 5 (CCR5). Initially we noted that vectors encoding CCR5 shRNA showed >30-fold lower viral titers than those of the empty vector. Co-transfection of expression plasmids encoding CCR5 in the producer cells yielded a tenfold increase in viral titer, thereby indicating that CCR5 mRNA, rather than HIV vector mRNA, could be the target of CCR5 shRNA. Similar increases in vector titer were observed after the H1 promoter was deleted. When Nodamura-virus B2 protein or Adenovirus VA.1 RNA (inhibitors of the Dicer-dependent siRNA pathway) were added to the producer cells, the vector titer rose almost to the level of that of the empty vector. Near identical increases in titer were observed with siRNA specifically directed against Dicer. Quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) suggested that the effects were in part caused by reduction in vector RNA in the producer cells. Similar results were observed with a retroviral vector. These results suggest that retrovirally-encoded shRNAs reduce vector titer in the producer cells through a Dicer-dependent mechanism which, to a large extent, can be reversed by inhibiting that pathway. This may have important implications for large-scale production of RNA vectors encoding shRNAs.
编码短发夹RNA（shRNA）的基因转移载体在解读基因功能方面很有用，并且正被考虑用于人类靶基因的治疗性敲低。我们构建了基于HIV的载体，其编码针对HIV共受体趋化因子（C-C基序）受体5（CCR5）的shRNA。最初我们注意到，编码CCR5 shRNA的载体的病毒滴度比空载体低30倍以上。在生产细胞中共转染编码CCR5的表达质粒使病毒滴度提高了10倍，从而表明CCR5 mRNA而非HIV载体mRNA可能是CCR5 shRNA的靶标。在删除H1启动子后，观察到载体滴度有类似的增加。当将诺达木病毒B2蛋白或腺病毒VA.1 RNA（Dicer依赖性siRNA途径的抑制剂）添加到生产细胞中时，载体滴度几乎上升到空载体的水平。用特异性针对Dicer的siRNA观察到滴度有几乎相同的增加。定量逆转录聚合酶链反应（RT-PCR）表明，这些效应部分是由生产细胞中载体RNA的减少引起的。用逆转录病毒载体也观察到了类似的结果。这些结果表明，逆转录病毒编码的shRNAs通过一种Dicer依赖性机制降低生产细胞中的载体滴度，在很大程度上，抑制该途径可以逆转这种机制。这可能对大规模生产编码shRNAs的RNA载体具有重要意义。
