HPB O02 Novel methods to modulate Nrf2 in liver regeneration

Abstract Background The unique regenerative capacity of the liver permits curative surgery for patients with malignancy. However, extensive resections a small future remnant risks post hepatectomy failure. Attempts to stimulate pre-resection regeneration have been refined over last two decades. Techniques such as portal vein ligation/embolization, hepatic embolization and ALPPS are in clinical use. All these interventions aim promote via subtle anti-oxidative response contralateral lobe. anti-oxidant transcription factor, nuclear erythoid 2-related factor 2 (Nrf2), has strongly linked enhancing regeneration. Furthermore, modulating Nrf2 pharmacologically shown enhance regeneration, both structurally functionally, animal models partial hepatectomy. Several activators (bardoxolone methyl (CDDO-Me), dimethyl fumarate, sulphoraphane) various phases trials, but each potential unwanted off-target effects. One method circumvent effects would be use gene-specific therapy. Small activating RNA (saRNA), is novel therapy using molecules upregulate specific gene. Here we present identification lead saRNA candidate Nrf2. Methods We obtained several human mouse saRNAs, different sequences, all designed target Human was screened hepatoma cell line (HepG2) stably transfected firefly A luciferase assay used determine expression. CDDO-Me positive control, transfection excluding (mock transfection) negative control. To ensure specificity candidates, further mutated versions were tested similar method. Endogenous gene expression measured quantitative PCR.Identification completed view vivo testing. Murine colorectal cancer (CT26) cells saRNA. Mutated samples ideal candidates fashion. Gene performed by qPCR. Similar controls used. Continuous data assessed normality Shapiro-Wilks test, analysed ANOVA (analysis variance) Tukey multiple comparisons test normally distributed data. Skewed Kruskal-Wallis’ test. p value <0.05 considered significant. Results Initial screening 11 saRNAs yielded PR91b PR145, 1.8 2.4 fold (p<0.0001) increase compared mock transfection. Specificity which revealed primers Nrf2, NQO1, GCLC GCLM (downstream targets) PR145 upregulated 1.5- 1.4-fold 1.8- 1.9-fold 2.2- 2.0-fold GCLM, respectively. showed minimal no upregulation or its downstream targets (p<0.001). Time course dose studies optimise protocol demonstrated an optimal final concentration 10nM period 4 days. qPCR 1 candidate, PR60, 3.0- 2.5-fold genes NQO1 NQO1. Conclusions date, there only one activator licensed Medicines Healthcare products Regulatory Agency – fumarate relapsing-remitting sclerosis. Ongoing trials exist aiming utilise related diseases well other non-hepatic (such treatment diabetic nephropathy COPD, amongst others). inherent problem drugs In this study demonstrate activation. Whilst not upregulating same degree vitro, benefit improved specificity, thereby eliminating off