Bifunctional Short Hairpin RNA (bi-shRNA): Design and Pathway to Clinical Application

Abstract

The discovery of RNA interference (RNAi) engendered great excitement and raised expectations regarding its potential applications in biomedical research and clinical usage. Over the ensuing years, expanded understanding of RNAi and preliminary results from early clinical trials tempered enthusiasm with realistic appraisal resulting in cautious optimism and a better understanding of necessary research and clinical directions. As a result, data from more recent trials are beginning to show encouraging positive clinical outcomes. The capability of delivering a pharmacologically effective dose to the target site while avoiding adverse host reactions still remains a challenge although the delivery technology continues to improve. We have developed a novel vector-driven bifunctional short hairpin RNA (bi-shRNA) technology that harnesses both cleavage-dependent and cleavage-independent RISC loading pathways to enhance knockdown potency. Consequent advantages provided by the bi-shRNA include a lower effective systemic dose than comparator siRNA/shRNA to minimize the potential for off-target side effects, due to its ability to induce both a rapid (inhibition of protein translation) and delayed (mRNA cleavage and degradation) targeting effect depending on protein and mRNA kinetics, and a longer duration of effectiveness for clinical applications. Here, we provide an overview of key molecular methods for the design, construction, quality control, and application of bi-shRNA that we believe will be useful for others interested in utilizing this technology.

Effect of a Diet Supplemented with the Moringa oleifera Seed Powder on the Performance, Egg Quality, and Gene Expression in Japanese Laying Quail under Heat-Stress

“…The housekeeping gene β-actin was used as the normalization control. Relative mRNA expression was calculated using the 2 (− ct) method, and the results were reported as a fold change [33].…”

KRAS mutant allele-specific expression knockdown in pancreatic cancer model with systemically delivered bi-shRNA KRAS lipoplex

“…The bi-shRNAs were designed according to previously published protocols [ 16 ]. The bi-shRNAs expression units were constructed by gene-synthesis method, the synthetic DNA was sequence confirmed both before and after (Epoch Life Sciences, Missouri City, TX) uni-directional insertion into the Sal I and Not I sites of the pUMVC3 mammalian expression vector.…”

Noncoding RNA therapeutics — challenges and potential solutions

“…Furin inhibition results in downregulation of immunosuppressive cytokines TGFβ1 and TGFβ2, thereby supporting T cell responses in the tumour. Bifunctional shRNAs cause both mRNA degradation and inhibition of translation, causing stronger and prolonged RNAi effects and allowing lower dosing to reduce off-targeting 191 . TGFβ1 and TGFβ2 were inhibited 90–100% in bi-shRNA furin-treated cells 189 , and treatment of patients with advanced cancer with Vigil caused an increase in circulating activated T cells and significantly improved survival 189 , 190 .…”

“…ShRNAs are traditionally introduced into cells using viral vector systems such as adenovirus-associated viruses, retroviruses or lentiviruses. Bifunctional shRNAs have higher knock-down efficacy as they simultaneously produce transcripts with perfect and imperfect complementarity to trigger degradation as well as translational inhibition 191 . Two liposomally delivered bifunctional shRNA constructs are currently being tested in phase I clinical trials: pbi-shRNA EWS/FLI1 (ref.…”