Anjaneyulu, M

Blakey, D

Habib, N

Habib, R

Huber, H

Jayaprakash, A

Mallappa, O

Reebye, V

Roberts, T

Rossi, J

Saetrom, P

Voutila, J

Molecular Therapy

Spiral - Imperial College Digital Repository

Mechanism And In Vivo Activity Of A Small Activating RNA Targeting CEBPA, A Novel Therapeutic In Clinical Trials For Liver Disease Jon Voutila1, Vikash Reebye2, Tom Roberts3, David Blakey1, Robert Habib1, Onkaramurthy Mallappa4, Muragundla Anjaneyulu4, Aravindakshan Jayaprakash4, Hans Huber5, Pal Saetrom6, John Rossi7, Nagy Habib1 1MiNA Therapeutics Ltd, 2Imperial College London, 3The Scripps Research Institute, 4Syngene Ltd, 5BioTD Strategies LLC, 6Norwegian University of Science and Technology, 7Beckman Research Institute of City of Hope   Small activating RNAs (saRNAs) are short double-stranded oligonucleotides that are designed to selectively increase gene transcription. Previously we designed an saRNA that upregulates the transcription factor CCATT/enhancer binding protein alpha (C/EBP-α) (Reebye et al., Hepatology 2014). We have now developed this saRNA into a clinical candidate, CEBPA-51, and here we investigate its mode of action and activity in an in vivo rat model of liver failure.  A nucleotide walk performed around bioinformatically-derived hotspots of saRNA activity in the CEBPA gene identified the sequence for CEBPA-51. This saRNA upregulates CEBPA mRNA 2.5-fold and C/EBP-α target gene albumin by 2.3-fold in human hepatocellular carcinoma HepG2 cells. A nuclear run-on assay confirmed that this is a transcriptionally-driven process. Mechanistic experiments demonstrate that Ago2 is required for saRNA activity, with the guide strand of the saRNA duplex shown to be associated with Ago2 and localized at the CEBPA genomic locus using RNA ChIP assays. Mutations in the seed sequence of CEBPA-51 caused a loss of activation, supporting a sequence-specific on-target saRNA activity of CEBPA-51. CEBPA-51 has been formulated in SMARTICLES® nanoparticles (MTL-CEBPA) for liver delivery and when administered at 4 mg/kg over 2 weeks leads to 90% inhibition of tumor growth in a diethylnitrosamine-induced cirrhotic liver cancer model and improvement in liver function. This novel drug is currently in a Phase I clinical trial for patients with liver cancer, and this represents the first human study of a saRNA therapeutic.  To investigate the activity of MTL-CEBPA in a model of fibrosis and acute liver failure, rats were exposed to CCl4 for up to 35 weeks. During this time animals were treated with either short term (2 weeks) or long term (14 weeks) MTL-CEBPA starting at week 8. We observed a dramatic improvement in liver function with restoration of AST, ALT, ammonia, and prothrombin time to near normal levels after just 2 weeks of MTL-CEBPA treatment. This was accompanied by highly significant reduction in hydroxyproline levels and other markers of liver fibrosis/inflammation. CEBPA mRNA expression in the liver as well as serum albumin were significantly elevated, demonstrating target engagement. At week 35, a significant increase in survival was observed where only 2 of the 9 rats deceased in the MTL-CEBPA group. No animals survived in the control group. These data strongly support exploring the additional clinical development of MTL-CEBPA for liver cirrhosis.  

English

Mechanism and in vivo activity of a small activating RNA targeting CEBPA, a novel therapeutic in clinical trials for liver disease

Mechanism and in vivo activity of a small activating RNA targeting CEBPA, a novel therapeutic in clinical trials for liver disease

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