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Preclinical Studies of the First in Human Sarna Drug Candidate for Liver Cancer

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Preclinical Studies of the First in Human Sarna Drug Candidate for Liver Cancer Oncogene (2018) 37:3216–3228 https://doi.org/10.1038/s41388-018-0126-2 ARTICLE Gene activation of CEBPA using saRNA: preclinical studies of the first in human saRNA drug candidate for liver cancer 1 2 2 3 4 1 Vikash Reebye ● Kai-Wen Huang ● Vivian Lin ● Sheba Jarvis ● Pedro Cutilas ● Stephanie Dorman ● 5 1 5 6,7 1 1 Simona Ciriello ● Pinelopi Andrikakou ● Jon Voutila ● Pal Saetrom ● Paul J. Mintz ● Isabella Reccia ● 8 9 5 10 5 1 John J. Rossi ● Hans Huber ● Robert Habib ● Nikos Kostomitsopoulos ● David C. Blakey ● Nagy A. Habib Received: 2 July 2017 / Revised: 2 December 2017 / Accepted: 12 December 2017 / Published online: 7 March 2018 © The Author(s) 2018. This article is published with open access Abstract Liver diseases are a growing epidemic worldwide. If unresolved, liver fibrosis develops and can lead to cirrhosis and clinical decompensation. Around 5% of cirrhotic liver diseased patients develop hepatocellular carcinoma (HCC), which in its advanced stages has limited therapeutic options and negative survival outcomes. CEPBA is a master regulator of hepatic function where its expression is known to be suppressed in many forms of liver disease including HCC. Injection of MTL-CEBPA, a small activating RNA oligonucleotide therapy (CEBPA-51) formulated in liposomal nanoparticles 1234567890();,: 1234567890();,: (NOV340- SMARTICLES) upregulates hepatic CEBPA expression. Here we show how MTL-CEBPA therapy promotes disease reversal in rodent models of cirrhosis, fibrosis, hepatosteatosis, and significantly reduces tumor burden in cirrhotic HCC. Restoration of liver function markers were observed in a carbon-tetrachloride-induced rat model of fibrosis following 2 weeks of MTL-CEBPA therapy. At 14 weeks, animals showed reduction in ascites and enhanced survival rates. MTL- CEBPA reversed changes associated with hepatosteatosis in non-alcoholic methionine and cholic-deficient diet-induced steaotic liver disease. In diethylnitrosamine induced cirrhotic HCC rats, MTL-CEBPA treatment led to a significant reduction in tumor burden. The data included here and the rapid adoption of MTL-CEBPA into a Phase 1 study may lead to new therapeutic oligonucleotides for undruggable diseases. Introduction Liver disease is caused by a multitude of components including obesity, alcoholism, and viral hepatitis. These synergistically culminate in an asymptomatic “compensated Electronic supplementary material The online version of this article cirrhosis” stage where the average life expectancy of (https://doi.org/10.1038/s41388-018-0126-2) contains supplementary patients is 10–13 years. This is usually followed by a material, which is available to authorized users. * Vikash Reebye 5 MiNA Therapeutics Ltd, London, UK [email protected] 6 Nagy A. Habib Department of Cancer Research and Molecular Medicine, [email protected] Norwegian University of Science and Technology, Trondheim, Norway 1 Department of Surgery, Imperial College London, London, UK 7 Department of Computer and Information Science, Norwegian University of Science and Technology, Trondheim, Norway 2 Department of Surgery and Hepatitis Research and Graduate Institute of Clinical Medicine, National Taiwan University, 8 Department of Molecular and Cellular Biology, Beckman Taipei, Taiwan Research Institute, City of Hope, Duarte, CA, USA 3 Department of Surgery and Cancer, Imperial College London, 9 BioTD Strategies, LLC, Lansdale, PA, USA London, UK 10 Biomedical Research Foundation of the Academy of Athens, 4 Cell Signalling and Proteomics Group, Centre for Haemato- Centre of Clinical, Experimental Surgery and Translational Oncology, Barts Cancer Institute, Queen Mary University of Research, Athens, Greece London, London, UK Gene activation of CEBPA using saRNA: preclinical studies of the first in human saRNA drug candidate. 3217 rapidly progressive phase of liver dysfunction termed The potential benefits of increasing expression levels of “decompensated cirrhosis” where the average life C/EBP-α have been considered in the literature to date. expectancy is reduced to as low as 2 years [1]. Downregulation of CEBPA is well known across many In the United States, there were 658,000 hospitalization different liver disease models including fibrosis and associated with cirrhosis in 2011, which culminated in $9.8 cirrhosis [7, 8]. Furthermore the importance of billion dollars in cost [2]. Despite recent medical advances, CEBPA activity in normal liver function is observed in liver cirrhosis remains a poorly treated stage of liver disease “old age”-related liver disease models, where hyperpho- and in 2013 claimed 1.2 million lives globally. Liver cir- sphorylation of CEBPA at serine residue 193 diminished rhosis is now the 8th leading cause of death in the United transcriptional activity [9]. In transgenic CEBPA knock-out States and the 13th leading cause of death globally, with a models, animals appear to develop liver tumors at an 46% increase in worldwide mortality over the past two increased rate [10, 11]. decades [1]. In order to validate the therapeutic benefit of increasing Progression of cirrhosis can be accelerated by the CEBPA expression in liver disease, we developed a ther- development of other complications which include renal apeutic oligonucleotide (CEBPA-saRNA) for intravenous impairment, sepsis, increasing viral titre and development delivery using nanoparticles with high tropsim to the liver of hepatocellular carcinoma (HCC) [3]. According to long- [12]. The successful outcome of this study led us to further term outcome studies, the severity of liver fibrosis is the develop CEBPA-saRNA into a GMP grade manufactured most important predictor for patient outcome [4]. clinical candidate (CEBPA-51) with early indications to Unresolved fibrosis ultimately leads to a decompensated show high target specificity and reliance on Ago2 and liver where the only treatment at this stage is liver trans- components of the RNA-interacting transcriptional plantation. However, the scarcity of organ donors, coupled activating complex [13]. This therapeutically viable saRNA with the increasing incidence of chronic liver disease which has been encased in liposomal nanoparticles (SMARTI- is intractable to conventional therapeutic compounds, poses CLES) [14] for the first in-human trial to study the outcome a major threat for patient survival. Patients who progress to of targeting CEBPA activation using saRNAs in patients HCC end up with a severely compromised liver owing to with HCC (clinicaltrials.gov–NCT0271601). the natural progression of the disease, which decreases their Here, we report the preclinical data obtained from opportunity to meet eligibility for curative surgery, trans- treating multiple liver disease models with the new ther- arterial chemoembolization or sorafenib treatment. apeutic candidate MTL-CEBPA. We show how restoration An improvement of liver function would be expected to of CEBPA expression in the liver markedly reverses disease benefit the general condition and Child Pugh status of in fibrosis and steatosis models, whilst improving liver patients; their quality of life and more significantly allow function tests and ultimately increasing survival rates in them to re-enter eligibility criteria for surgical interventions, long-term fibrosis (CCl4- induced rats). The data included which would ultimately enhance chances of survival. here and the rapid adoption of MTL-CEBPA into a Phase A treatment that reverses fibrosis, improves liver 1 study may lead to new therapeutic oligonucleotides for function, whilst inhibiting tumor growth would be a major undruggable diseases. therapeutic breakthrough and here we present data for a novel RNA-based drug candidate in the form of small activating RNAs (saRNAs), which is currently in a phase I Results clinical trial for patients with advanced cirrhotic HCC (ClinicalTrials.gov–NCT0271601). In vitro activation of C/EBP-α by CEBPA-51 saRNAs differ from other RNA-based therapeutics in that they upregulate, rather than inhibit the expression of The ability of CEBPA-51 to increase CEBPA mRNA their therapeutic target [5]. The mechanism of action expression in vitro was demonstrated in murine (AML12); involves loading of saRNA into the Argonaute 2 (Ago2) rat (Clone 9) liver cell line, human (HEPG2) liver cells, and protein complex, translocation into the nucleus, binding to primary human hepatocytes, (Fig. 1a). In each case, trans- the genomic target locus and promoting increased fection of the newly GMP developed CEBPA-51 saRNA transcriptional activity by RNA Polymerase 2 [6, 13]. significantly increased CEBPA mRNA levels by about Here we report an saRNA therapy that was designed to twofold, whereas the non-specific control RNA oligonu- increase production of CEBPA (CCAAT/enhancer-binding cleotide (siFLUC) was inactive. In human primary protein alpha), a master transcriptional factor in the hepatocytes, transfection of CEBPA-51 showed increased liver, which resets the natural gene regulatory mechanism expression of CEBPA mRNA and significant changes to of hepatocytes to reduce fibrosis and reverse liver expression of downstream targets of CEBPA. These dysfunction. included key liver function factors including albumin, 3218 V. Reebye et al. Fig. 1 Transcriptional activity of CEBPA-51 in vitro. a CEBPA-51 (0.0033). c A western blot panel probed with antibody specificto saRNA transfection increases CEBPA mRNA in murine AML12, HNF4A (Abcam; ab41898), CYP3A4 (Abcam; ab155029), Albumin Rattus (Clone 9), Human (HEPG2) and human (Primary hepatocytes). (Abcam; ab106582), and
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