Liver Activation of Hepatocellular Nuclear Factor-4α by Small Activating RNA Rescues Dyslipidemia and Improves Metabolic Profile

Objective: Nonalcoholic fatty liver disease (NAFLD) culminates in insulin resistance and metabolic syndrome. As yet there is no approved single agent that targets steatosis or its pathological progression to hepatitis. Hepatocyte-nuclear-factor 4-alpha (HNF-4α) is at the centre of a complex transcriptional network where its disruption is directly linked to diabetes and steatosis. Re-activating HNF-4α expression in NAFLD could therefore reverse pathology. Methods: A small activating RNAs (saRNA), designed synthetically for upregulating HNF-4α expression in hepatocytes, was administered intravenously (I.V) into a rat NAFLD model. These animals were sustained on a high fat diet (HFD) for 16 weeks to induce pathology. I.V. delivery (3x injections) of saRNAs (0.6mg/kg) beginning at week 16 was performed using 5-(G5)- triethanolamine-core poly(amidoamine) dendrimers. Treatment continued for a further two weeks (1 x injection per week) whilst the animals were maintained on HFD.. Results: saRNA treatment caused a significant increase in HNF4A transcript levels in the liver. This resulted in a reduction in liver triglyceride and cholesterol; an increased HDL/LDL ratio and decreased white adipose tissue/body weight ratio. Since HNF-4α is a key transcriptional factor in hepatocytes we analysed saRNA transfected liver cells for global phoshoproteomic changes driven by HNF4A upregulation. We observed significant beneficial changes in proteins regulating sphingolipid metabolism, fatty acid β-oxidation and ketogenesis, detoxification of reactive oxygen species and lipid transport Conclusions: We demonstrate that HNF-4α can be specifically activated by saRNAs when delivered using nanoparticles with high tropism to the liver. HNF4A-saRNA treatment induced a favorable metabolic profile and represents a novel potential agent for the treatment of NAFLD. Funding Statement: This work was funded in part by Imperial College London; National Taiwan University College of Medicine and MiNA Therapeutics. Declaration of Interests: N.A.H, J.R, R.H, J.N, D.C.B and V.R have equity in MiNA Therapeutics Limited. All remaining authors have no conflicts to declare. Ethics Approval Statement: This study was approved by the University of Taiwan animal welfare committee.