Delivery of RNAi Therapeutics to the Airways—From Bench to Bedside

Qiu, Yingshan; Lam, Jenny K. W.; Leung, Susan W. S.; Liang, Wanling

shRNA is a sequence of RNA transcribed in the nucleus of the cells from a DNA vector by either RNA polymerase II or III. The primary transcript is called primary shRNA (pri-shRNA), which contains a hairpin like stem-loop structure. The pri-shRNA is processed into a 50-70 nucleotides long loop-stem precursor shRNA (pre-shRNA) by a protein complex containing the RNase III nuclease Drosha and the dsRNA binding domain protein DGCR8. It is then transported to the cytoplasm through a specialized nuclear membrane protein, exportin-5 (Exp5). The loop sequence of the pre-shRNA is cleaved by the Dicer to form a double-stranded siRNA. This endogenously produced siRNA is loaded into RISC and induce RNAi through the similar process as the synthetic siRNA [18, 19] . Since shRNA expression unit can be incorporated into viral vectors and continuously synthesized by the host cell, it can induce long-lasting gene silencing effect. The RISC-loading process of shRNA is about ten times more efficient than siRNA, implicating that lower dose of shRNA is required to maintain the therapeutic efficacy with less off-target effect [20] . However, the shRNA approach is a DNA-based strategy depending on the expression of shRNA encoding gene which often requires viral vectors. From the delivery perspective, the introduction of synthetic siRNA to the cytoplasm is a more straightforward way to induce RNAi. The use of viral vectors for delivery poses safety concerns in its therapeutic applications [15] .