ASCO 2018

Background: MTL-CEBPA is a liposomal formulation of saRNA targeting the transcription factor C/EBP-α, which acts as a master regulator of liver homeostasis and multiple oncogenic processes including cell cycle control, proliferation and angiogenesis and inhibits hepatocellular cancer (HCC) tumor growth in preclinical models. MTL-CEBPA is the first saRNA and the first drug targeting C/EBP-α entering clinical trials.

Methods: Patients (pts) with advanced HCC (Child-Pugh A/B) or secondary liver cancer, were enrolled in a 3+3 dose escalation study. MTL-CEBPA is administered as a 1-hr IV infusion on Day 1, 8 and 15 of a 28 day cycle. The primary endpoint was safety and the secondary endpoints included PK, liver function improvement and anti-tumor activity. Correlative studies include C/EBP-α mRNA levels in PBMCs and tumor tissue, evaluation of C/EBP-α downstream target genes (e.g.TGFβ) and distal target engagement in WBCs (e.g.IL-6, NF-κB).

Results: 19 participants have been treated across 5 dose levels (28-130 mg/m²): 13M/6F, median age 67 yrs (range 27 – 80), ECOG PS 0/1: 9/10. Tumour types include HCC (13), colorectal (4) and fibrolamellar (2). The most common treatment-related AEs (all grades/grade 3) include fatigue (9/1), diarrhoea (5/0), AST increase (5/1), low platelets (2/1) hyperbilirubinaemia (5/1) and hypophosphataemia (4/1). Maximum tolerated dose has not yet been reached. Serum PK analysis shows a terminal half life of > 24 hrs, with dose proportional C_max and AUC. Analysis of WBCs showed a significant increase of C/EBP-α expression during treatment providing evidence of target engagement. Of 10 evaluable pts with HCC, 4 pts have had SD≥ 4months, with one patient having an ongoing PR for 18 months associated with 73% decrease in tumour volume and reduction in IL-6, NF-κB and IFN-γ.

Conclusions: Once weekly MTL-CEBPA therapy was well tolerated, shows promising PD and initial clinical response in patients with advanced HCC. Updated results for the dose escalation will be presented. Clinical trial information: NCT02716012

Oncogene vol 37, pp3216–3228 (2018)

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 (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.

Molecular Therapy Vol. 25 No 12 December 2017

Small activating RNAs (saRNAs) are short double-stranded oligonucleotides that selectively increase gene transcription. Here, we describe the development of an saRNA that upregulates the transcription factor CCATT/enhancer binding protein alpha (CEBPA), investigate its mode of action, and describe its development into a clinical candidate. A bioinformatically directed nucleotide walk around the CEBPA gene identified an saRNA sequence that upregulates CEBPA mRNA 2.5-fold in human hepatocellular carcinoma cells.

A nuclear run-on assay confirmed that this upregulation is a transcriptionally driven process. Mechanistic experiments demonstrate that Argonaute-2 (Ago2) is required for saRNA activity, with the guide strand of the saRNA shown to be associated with Ago2 and localized at the CEBPA genomic locus using RNA chromatin immunoprecipitation (ChIP) assays. The data support a sequence-specific on-target saRNA activity that leads to enhanced CEBPA mRNA transcription. Chemical modifications were introduced in the saRNA duplex to prevent activation of the innate immunity.

This modified saRNA retains activation of CEBPA mRNA and downstream targets and inhibits growth of liver cancer cell lines in vitro. This novel drug has been encapsulated in a liposomal formulation for liver delivery, is currently in a phase I clinical trial for patients with liver cancer, and represents the first human study of an saRNA therapeutic.

AASLD 2016

We have developed a small activating RNA, CEBPA-51, that upregulates the transcription factor CCATT/enhancer binding protein alpha (C/EBP-α) and formulated it in SMARTICLES® (MTL-CEBPA) for liver delivery. MTL-CEBPA is currently in clinical trials in patients with liver cancer. Here we show long term treatment of MTL-CEBPA improves survival in a CCl4 model of acute liver failure.

Liver fibrosis/acute failure was induced in Sprague Dawley rats by i.p. injection of CCl4 for 28 weeks. At week 8, rats (n=9) were treated with twice weekly i.v. of PBS or 1mg/kg MTL-CEBPA for 14 weeks. Liver function parameters and the degree of ascites and overall survival was monitored.

After 2 weeks of MTL-CEBPA treatment, serum albumin (a direct target of C/EBP-α) significantly increased (p<0.05) and bilirubin levels significantly decreased (p<0.001) when compared to PBS. Liver toxicity and function: AST (50%), ALT (57%), prothrombin time (17%) and ammonia (42%) all showed significant improvement (% values) by week 13 when compared to PBS. All effects were maintained for 28 weeks. After 14 weeks of CCl4 treatment 8/9 of the PBS group animals had very significant ascites compared to only 2/9 in the MTL-CEBPA treatment animals. In terms of overall survival, by week 28, 6/9 animals in the PBS group were deceased vs just 1/9 animals in the MTL-CEBPA treated animals (p<0.01).

Conclusion: MTL-CEBPA increases serum albumin and decreases serum bilirubin. This was accompanied by a significant reduction in serum liver enzymes and improvement in liver function. The levels of ascites were dramatically reduced and a significant improvement in overall survival was seen post treatment with MTL-CEBPA. These data strongly support exploring the clinical development of MTL-CEBPA in liver failure.

Perspectives on Current Science – June 2016

Despite the fact that the RNAa mechanism remained unclear it took only ten years between first publication and first application to the clinic (see MiNA Therapeutics). How ironic then that two recent papers have finally shed some more light on how exactly RNAa works.

EASL Liver Fibrosis 2016

Introduction: Chronic liver disease is a growing epidemic worldwide responsible for progressive liver fibrosis and liver failure. CCAAT/ enhancer binding protein alpha (C/EBPα) is one of the master regulators in the liver for normal differentiation and metabolic function. Its attenuation is frequently observed in liver disease.

Aims: We have evaluated three different models of chronic liver disease to determine if therapeutic activation of CEBPA, achieved by intravenous delivery of a small activating RNA (saRNA) to CEBPA encapsulated in NOV340 SMARTICLES® (MTL-CEBPA), would reverse liver failure.

Results: Non-alcoholic steatohepatitis was induced in C57BL/6 mice with methionine choline deficient diet (MCD). Upon administration of MTL-CEBPA (0.3, 1 and 3mg/kg) we observed a significant 55% reduction in staining of alpha smooth muscle actin compared to control. In addition we saw significant reduction of serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) to near normal levels and significant 70% reduction in liver triglyceride and hepatic lipid accumulation at all dose range. To induce hepatic fibrosis, wild-type Sprague Dawley (SD) rats were treated for 10 weeks with Carbon tetrachloride (CCl4). Injury to the liver were confirmed by Sirius red and Masson’s trichrome staining. AST, ALT and hydroxyrproline showed significant reversal to near normal levels after 2 weeks of MTL-CEBPA treatment (4 doses of 3.0 mg/kg). Moreover, mortality significantly decreased (11% in MTL-CEBPA treated groups vs 44% in control). An acute liver failure model was induced in SD rats subjected to 350mg/kg of Thioacetamide (TAA) intraperitoneally. Similar to the above models a significant improvement in all liver parameters measured including ALT, AST and bilirubin were observed following a single dose of MTL-CEBPA injection.

Conclusions: These studies demonstrated the crucial role of C/EBPα in maintaining normal liver function and highlight the potential of using MTL-CEBPA to activate CEBPA as a treatment of liver fibrosis and other liver disease.

PLoS ONE 11(4): e0153117

Hepatocellular carcinoma is associated with high mortality, and tumor metastasis is an important reason for poor prognosis. However, metastasis has not been effectively prevented in clinical therapy and the mechanisms underlying metastasis have not been fully characterized. CCAAT/enhancer-binding protein-α (C/EBPα) is a transcriptional regulator with an essential role in tumor metastasis. We used short-activating RNAs (saRNA) to enhance expression of C/EBPα. Intravenous injection of C/EBPα-saRNA in a nude mouse liver orthotopic xenograft tumor model inhibited intrahepatic and distant metastasis. C/EBPα-saRNA-treated mice showed increased serum levels of albumin and decreased alanine aminotransferase (ALT), glutamic-oxalacetic transaminase (AST), indicating a role of C/EBPα in improving liver function. Migration and invasion were inhibited in hepatoma cell lines transfected with C/EBPα-saRNA. We also observed an inhibition of epithelial-mesenchymal transition (EMT) and suppression of epidermal growth factor receptor (EGFR), EGFR phosphorylation, and β-catenin in C/EBPa-saRNA-transfected cells. Our results suggested that C/EBPα-saRNA successfully inhibited HCC metastasis by inhibiting EGFR/β-catenin signaling pathway mediated EMT in vitro and in vivo.

Cell Research (2016): 1-16. doi:10.1038/cr.2016.22

Small activating RNAs (saRNAs) targeting specific promoter regions are able to stimulate gene expression at the transcriptional level, a phenomenon known as RNA activation (RNAa). It is known that RNAa depends on Ago2 and is associated with epigenetic changes at the target promoters. However, the precise molecular mechanism of RNAa remains elusive. Using human CDKN1A (p21) as a model gene, we characterized the molecular nature of RNAa. We show that saRNAs guide Ago2 to and associate with target promoters. saRNA-loaded Ago2 facilitates the assembly of an RNA-induced transcriptional activation (RITA) complex, which, in addition to saRNA-Ago2 complex, includes RHA and CTR9, the latter being a component of the PAF1 complex. RITA interacts with RNA polymerase II to stimulate transcription initiation and productive elongation, accompanied by monoubiquitination of histone 2B. Our results establish the existence of a cellular RNA-guided genome-targeting and transcriptional activation mechanism and provide important new mechanistic insights into the RNAa process.

Nucleic Acids Research, 2016 1 doi: 10.1093/nar/gkw076

RNA activation (RNAa) is the upregulation of gene expression by small activating RNAs (saRNAs). In order to investigate the mechanism by which saRNAs act in RNAa, we used the progesterone receptor (PR) gene as a model, established a panel of effective saRNAs and assessed the involvement of the sense and antisense strands of saRNA in RNAa. All active saRNAs had their antisense strand effectively incorporated into Ago2, whereas such consistency did not occur for the sense strand. Using a distal hotspot for saRNA targeting at 1.6-kb upstream from the PR transcription start site, we further established that gene activation mediated by saRNA depended on the complementarity of the 5′ region of the antisense strand, and that such activity was largely abolished by mutations in this region of the saRNA. We found markedly reduced RNAa effects when we created mutations in the genomic target site of saRNA PR-1611, thus providing evidence that RNAa depends on the integrity of the DNA target. We further demonstrated that this saRNA bound the target site on promoter DNA. These results demonstrated that saRNAs work via an on-site mechanism by binding to target genomic DNA in a seed-region-dependent manner, reminiscent of miRNA-like target recognition.

AASLD 2015

The transcription factor CCATT/enhancer binding protein alpha (C/EBP-α) is known to have an important regulatory role in the maintenance of normal hepatocyte function and response to injury. We developed a small activating RNA (saRNA) that was demonstrated to significantly up-regulate C/EBP-α expression in primary hepatocytes.

This saRNA was subsequently encapsulated in an anionic liposome as a development candidate for clinical use (MTL-CEBPA). Liver failure was induced in Sprague Dawley rats by twice weekly i.p. injection of CCl4 for a duration of 8 weeks. For a further 2 weeks of the CCl4 regiment, animals were treated by twice weekly i.v. injection of MTL-CEBPA via tail vein at 0.3mg/ kg, 1mg/kg and 3mg/kg. We demonstrate reversal and near normalisation of clinically relevant parameters following all treatment doses of MTL-CEBPA including, at 3mg/kg, bilirubin (64% decrease), circulating alanine and aspartate aminotransferase (59% and 62% decrease respectively) and prothrombin time (19% decrease).

We also observed a significant increase in serum albumin and total protein as well as a significant decrease in alkaline phosphatase and gamma-glutamyl-transpeptidase. Liver hydroxyproline significantly decreased in a dose-dependent manner in addition to a significant increase in body weight with no associated toxicity. Here we present a novel development candidate, MTL-CEBPA, that safely up-regulates C/EBP-α, as a potential treatment for liver failure in vivo. Clinical studies with MTL-CEBPA are expected to begin in early 2016.

Science-Business eXchange 7, doi:10.1038/scibx.2014.136

Although siRNAs have been validated as therapeutics to knock down gene expression, short double-stranded RNAs can also turn on gene expression. An academic team sponsored by MiNA Therapeutics has attacked liver cancer in rats by using a short activating RNA to upregulate expression of a tumor suppressor.

Hepatology, 59: 216–227. doi: 10.1002/hep.26669

Hepatocellular carcinoma (HCC) occurs predominantly in patients with liver cirrhosis. Here we show an innovative RNA-based targeted approach to enhance endogenous albumin production while reducing liver tumor burden. We designed short-activating RNAs (saRNA) to enhance expression of C/EBPα (CCAAT/enhancer-binding protein-α), a transcriptional regulator and activator of albumin gene expression. Increased levels of both C/EBPα and albumin mRNA in addition to a 3-fold increase in albumin secretion and 50% decrease in cell proliferation was observed in C/EBPα-saRNA transfected HepG2 cells. Intravenous injection of C/EBPα-saRNA in a cirrhotic rat model with multifocal liver tumors increased circulating serum albumin by over 30%, showing evidence of improved liver function. Tumor burden decreased by 80% (P = 0.003) with a 40% reduction in a marker of preneoplastic transformation. Since C/EBPα has known antiproliferative activities by way of retinoblastoma, p21, and cyclins, we used messenger RNA (mRNA) expression liver cancer-specific microarray in C/EBPα-saRNA-transfected HepG2 cells to confirm down-regulation of genes strongly enriched for negative regulation of apoptosis, angiogenesis, and metastasis. Up-regulated genes were enriched for tumor suppressors and positive regulators of cell differentiation. A quantitative polymerase chain reaction (PCR) and western blot analysis of C/EBPα-saRNA-transfected cells suggested that in addition to the known antiproliferative targets of C/EBPα, we also observed suppression of interleukin (IL)6R, c-Myc, and reduced STAT3 phosphorylation. Conclusion: A novel injectable saRNA-oligonucleotide that enhances C/EBPα expression successfully reduces tumor burden and simultaneously improves liver function in a clinically relevant liver cirrhosis/HCC model.

Molecular Therapy Nucleic Acids (2013) 2, e97; doi:10.1038/mtna.2013.23

Upon functional loss of insulin producing islet β-cells, some patients with diabetes become dependent on life-long insulin supplementation therapy. Bioengineering surrogate insulin producing cells is an alternative replacement strategy. We have developed a novel approach using short-activating RNA oligonucleotides to differentiate adult human CD34+ cells into insulin-secreting cells. By transfecting RNA to increase transcript levels of the master regulator of insulin biosynthesis, v-maf musculoaponeurotic fibrosarcoma oncogene homolog A (MafA), several pancreatic endodermal genes were upregulated during the differentiation procedure. These included Pancreatic and duodenal homeobox gene-1 (PDX1), Neurogenin 3, NeuroD, and NK6 homeobox 1 (NKx6-1). Differentiated CD34+ cells also expressed glucokinase, glucagon-like peptide 1 receptor (GLP1R), sulfonylurea receptor-1 (SUR1) and phogrin—all essential for glucose sensitivity and insulin secretion. The differentiated cells appropriately processed C-peptide and insulin in response to increasing glucose stimulation as shown by enzyme-linked immunosorbent assay (ELISA), fluorescence-activated cell sorting analysis, western blotting, and immunofluorescence staining. We provide a new approach using short-activating RNA in developing insulin producing surrogate cells for treating diabetes.

Molecular Therapy Nucleic Acids (2012) 1, e35; doi:10.1038/mtna.2012.20

It is now recognized that small noncoding RNA sequences have the ability to mediate transcriptional activation of specific target genes in human cells. Using bioinformatics analysis and functional screening, we screened short-activating RNA (saRNA) oligonucleotides designed to target the promoter regions of the pluripotency reprogramming factors, Kruppel-like factor 4 (KLF4) and c-MYC. We identified KLF4 and c-MYC promoter-targeted saRNA sequences that consistently induced increases in their respective levels of nascent mRNA and protein expression in a time- and dose-dependent manner, as compared with scrambled sequence control oligonucleotides. The functional consequences of saRNA-induced activation of each targeted reprogramming factor were then characterized by comprehensively profiling changes in gene expression by microarray analysis, which revealed significant increases in mRNA levels of their respective downstream pathway genes. Notably, the microarray profile after saRNA-mediated induction of endogenous KLF4 and c-MYC showed similar gene expression patterns for stem cell- and cell cycle-related genes as compared with lentiviral vector-mediated overexpression of exogenous KLF4 and c-MYC transgenes, while divergent gene expression patterns common to viral vector-mediated transgene delivery were also noted. The use of promoter-targeted saRNAs for the activation of pluripotency reprogramming factors could have broad implications for stem cell research.

Nature Chemical Biology 3, 166 – 173 (2007)

The ability to selectively activate or inhibit gene expression is fundamental to understanding complex cellular systems and developing therapeutics. Recent studies have demonstrated that duplex RNAs complementary to promoters within chromosomal DNA are potent gene silencing agents in mammalian cells. Here we report that chromosome-targeted RNAs also activate gene expression. We have identified multiple duplex RNAs complementary to the progesterone receptor (PR) promoter that increase expression of PR protein and RNA after transfection into cultured T47D or MCF7 human breast cancer cells. Upregulation of PR protein reduced expression of the downstream gene encoding cyclooygenase 2 but did not change concentrations of estrogen receptor, which demonstrates that activating RNAs can predictably manipulate physiologically relevant cellular pathways. Activation decreased over time and was sequence specific. Chromatin immunoprecipitation assays indicated that activation is accompanied by reduced acetylation at histones H3K9 and H3K14 and by increased di- and trimethylation at histone H3K4. These data show that, like proteins, hormones and small molecules, small duplex RNAs interact at promoters and can activate or repress gene expression.

Proceedings of the National Academy of Sciences of the United States of America Vol. 103, No. 46 (Nov. 14, 2006), pp. 17337-17342

Recent studies have shown that small noncoding RNAs, such as microRNAs and siRNAs, regulate gene expression at multiple levels including chromatin architecture, transcription, RNA editing, RNA stability, and translation. Each form of RNA-dependent regulation has been generally found to silence homologous sequences and collectively called RNAi. To further study the regulatory role of small RNAs at the transcriptional level, we designed and synthesized 21-nt dsRNAs targeting selected promoter regions of human genes E-cadherin, p21WAF1/CIP1 (p21), and VEGF. Surprisingly, transfection of these dsRNAs into human cell lines caused long-lasting and sequence-specific induction of targeted genes. dsRNA mutation studies reveal that the 5′ end of the antisense strand, or “seed” sequence, is critical for activity. Mechanistically, the dsRNA-induced gene activation requires the Argonaute 2 (Ago2) protein and is associated with a loss of lysine-9 methylation on histone 3 at dsRNA-target sites. In conclusion, we have identified several dsRNAs that activate gene expression by targeting noncoding regulatory regions in gene promoters. These findings reveal a more diverse role for small RNA molecules in the regulation of gene expression than previously recognized and identify a potential therapeutic use for dsRNA in targeted gene activation.