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.

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.