BAY11 enhances OCT4 synthetic mRNA expression in adult human skin cells

Abstract

IntroductionThe OCT4 transcription factor is involved in many cellular processes, including development, reprogramming, maintaining pluripotency and differentiation. Synthetic OCT4 mRNA was recently used (in conjunction with other reprogramming factors) to generate human induced pluripotent stem cells. Here, we discovered that BAY 11-7082 (BAY11), at least partially through an NF-κB-inhibition based mechanism, could significantly increase the expression of OCT4 following transfection of synthetic mRNA (synRNA) into adult human skin cells.MethodsWe tested various chemical and molecular small molecules on their ability to suppress the innate immune response seen upon synthetic mRNA transfection. Three molecules - B18R, BX795, and BAY11 - were used in immunocytochemical and proliferation-based assays. We also utilized global transcriptional meta-analysis coupled with quantitative PCR to identify relative gene expression downstream of OCT4.ResultsWe found that human skin cells cultured in the presence of BAY11 resulted in reproducible increased expression of OCT4 that did not inhibit normal cell proliferation. The increased levels of OCT4 resulted in significantly increased expression of genes downstream of OCT4, including the previously identified SPP1, DUSP4 and GADD45G, suggesting the expressed OCT4 was functional. We also discovered a novel OCT4 putative downstream target gene SLC16A9 which demonstrated significantly increased expression following elevation of OCT4 levels.ConclusionsFor the first time we have shown that small molecule-based stabilization of synthetic mRNA expression can be achieved with use of BAY11. This small molecule-based inhibition of innate immune responses and subsequent robust expression of transfected synthetic mRNAs may have multiple applications for future cell-based research and therapeutics.

Inhibition of the Inflammatory Pathway Enhances Both the <i>in Vitro</i> and <i>in Vivo</i> Transfection Activity of Exogenous <i>in Vitro</i>-Transcribed mRNAs Delivered by Lipid Nanoparticles

“…However, it was also reported that small molecule inhibitors of type-I interferon signaling showed no enhancing effects on the overall production of proteins in single transfection of IVT-mRNA. 12) On the other hand, Awe et al reported that inhibitors of nuclear factor-kappaB (NF-κB) signaling, BAY11-708 and BX795, significantly elevated the transfection efficiency of the IVT-mRNA. 13) Other possible targets are the proteins that are involved in integrated stress responses (ISRs), since cellular stresses including the recognition of exogenous RNA induce the suppression of canonical cap-dependent translation via the phosphorylation eIF2α .…”

Suppression of mRNA Nanoparticle Transfection in Human Fibroblasts by Selected Interferon Inhibiting Small Molecule Compounds

“…In this report, we hypothesize that active inhibition of PAMPs and/or the IFN activation pathway is also a reasonable strategy to enhance mRNA transfection. Indeed, this concept of active inhibition was recently reviewed [ 9 ] and a proof-of-principle study focused on the use of small molecules Bay11 and BX795 were reported [ 12 ]. Interestingly, many other small molecules that had been reported to inhibit IFN were also available commercially.…”

Corticosteroids and cellulose purification improve respectively the in vivo translation and vaccination efficacy of self-amplifying mRNAs

“…In contrast, Awe et al (2013) reported enhanced in vitro translation of the transcription factor OCT4 from a synthetic mRNA upon BAY11 supplementation [35]. However, the enhanced OCT4 translation was not achieved when the type I IFN decoy receptor B18R was supplemented, indicating that the observed increase in translation was independent of type I interferons [35].…”