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Excessive or inappropriate inflammatory responses can cause serious and even fatal diseases. The CCAAT/enhancer-binding protein alpha (CEBPA) gene encodes C/EBPα, a transcription factor that plays a fundamental role in controlling maturation of the myeloid lineage and is also expressed during the late phase of inflammatory responses when signs of inflammation are decreasing. MTL-CEBPA, a small activating RNA targeting for upregulation of C/EBPα, is currently being evaluated in a phase 1b trial for treatment of hepatocellular carcinoma. After dosing, subjects had reduced levels of pro-inflammatory cytokines, and we therefore hypothesized that MTL-CEBPA has anti-inflammatory potential. The current study was conducted to determine the effects of C/EBPα saRNA - CEBPA-51 - on inflammation in vitro and in vivo after endotoxin challenge. CEBPA-51 led to increased expression of the C/EBPα gene and inhibition of pro-inflammatory cytokines in THP-1 monocytes previously stimulated by E. coli-derived lipopolysaccharide (LPS). Treatment with MTL-CEBPA in an LPS-challenged humanized mouse model upregulated C/EBPα mRNA, increased neutrophils, and attenuated production of several key pro-inflammatory cytokines, including TNF-α, IL-6, IL-1β, and IFN-γ. In addition, a Luminex analysis of mouse serum revealed that MTL-CEBPA reduced pro-inflammatory cytokines and increased the anti-inflammatory cytokine IL-10. Collectively, the data support further investigation of MTL-CEBPA in acute and chronic inflammatory diseases where this mechanism has pathogenic importance., MTL-CEBPA is a small activating RNA that targets the upregulation of transcription factor C/EBPα, currently being evaluated in a phase 1b trial for treatment of hepatocellular carcinoma. Zhou et al. demonstrate that MTL-CEBPA increases the expression of C/EBPα and displays anti-inflammatory effects in a tissue culture system and a humanized mouse model.

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.