Enhanced target gene knockdown by a bifunctional shRNA: a novel approach of RNA interference

Abstract

RNA interference (RNAi) is a natural cellular regulatory process that inhibits gene expression by transcriptional, post-transcriptional and translational mechanisms. Synthetic approaches that emulate this process (small interfering RNA (siRNA), short hairpin RNA (shRNA)) have been shown to be similarly effective in this regard. We developed a novel 'bifunctional' RNAi strategy, which further optimizes target gene knockdown outcome. A bifunctional construct (bi-sh-STMN1) was generated against Stathmin1, a critical tubulin modulator that is overexpressed in human cancers. The bifunctional construct is postulated to concurrently repress the translation of the target mRNA (cleavage-independent, mRNA sequestration and degradation) and degrade (through RNase H-like cleavage) post-transcriptional mRNA through cleavage-dependent activities. Bi-sh-STMN1 showed enhanced potency and durability in parallel comparisons with conventional shRNA and siRNAs targeting the same sequence. Enhanced STMN1 protein knockdown by bi-sh-STMN1 was accompanied by target site cleavage at the mRNA level showed by the rapid amplification of complementary DNA ends (RACE) assay. Bi-sh-STMN1 also showed knockdown kinetics at the mRNA level consistent with its multieffector silencing mechanisms. The bifunctional shRNA is a highly effective and advantageous approach mediating RNAi at concentrations significantly lower than conventional shRNA or siRNA. These results support further evaluations.

Stathmin 1: a protein with many tasks. New biomarker and potential target in cancer

“…The latter may be due to the effect of modulation of microtubule network mobility on the proportion of Bax/Bcl-2 and Bax/Bcl-xL heterodimers [35,36]. More recently, we have demonstrated marked (93%) knockdown of STMN1 using a novel bifunctional RNA interference technology [37]. We have shown a 5-log dosing improvement in growth inhibition involving CCL-247 colon cancer cells comparing bi-shRNAi STMN1 to siRNAi STMN1 targeting the identical mRNA sequence and in coordination with cleavage product quantitation.…”

PDX1 associated therapy in translational medicine

“…The bifunctional shRNA consists of two stem-loop shRNA (shorthairpin RNA) structures: one cleavage-dependent unit with a perfectly matched passenger-strand and guide-strand, and one cleavage-independent unit composed of a mismatched double strand. It is able to induce both RNase-H like cleavage and non-cleavage mediated degradation of the target mRNA and inhibit translation concurrently, leading to more rapid onset of gene silencing, higher efficacy and greater durability (17,(20)(21)(22) (5,7). Intravenous infusions of bi-shRNA PDX1 nanoplexes in a large bio-relevant Yucatan mini-pig model were associated minimal toxicity (23).…”

Cancer targeting vaccines: Surrogate measures of activity

“…In order to further expand upon lessons learned for prior immune stimulating approaches, we tested a dual expressive vector containing human GMCSF DNA with a novel bifunctional RNA interference technology 7 targeting furin. Furin is a proprotein convertase which upregulates both TGFβ1 and TGFβ2, potent cancer produced immune inhibitors.…”