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.
Small activating RNAs (saRNAs) are short double-stranded oligonucleotides that are designed to selectively increase gene transcription and have great therapeutic potential. Previously we designed AW1-50, a saRNA that upregulates the transcription factor CCATT/enhancer binding protein alpha (CEBPA) (Reebye et al., Hepatology 2014). Here we investigate its mode of action and describe its optimization into a clinical candidate, CEBPA-51. A nucleotide walk was performed around hotspots in the CEBPA gene, previously selected by bioinformatic design. This identified AW1-51, a saRNA sequence in the AW1-50 hotspot region that showed enhanced activity, upregulating CEBPA mRNA 2.5-fold and CEBPA target gene albumin by 2.3-fold in human hepatocellular carcinoma HepG2 cells. The activation of CEBPA is dose-dependent with an EC50 of 5nM.
A nuclear run-on assay confirmed that the upregulation of CEBPA mRNA was a transcriptionally driven process. Further mechanistic experiments using Argonaute-2 (Ago2) KO cells demonstrated that Ago2 is required for saRNA activity. The guide strand of the saRNA duplex was shown to be associated with Ago2 and localized at the CEBPA genomic locus using RNA ChIP assays. Mutations in the seed sequence of AW1-51 caused a complete loss of saRNA activity, while mutations to prevent RNAi cleavage did not prevent gene activation. The data support a sequence-specific on-target saRNA activity of AW1-51 that leads to enhanced CEBPA mRNA transcription.
Chemical modifications were introduced in the AW1-51 saRNA duplex to prevent sequence-specific activation of the innate immune system. This final modified saRNA, CEBPA-51, retains activation of CEBPA mRNA and downstream targets, and inhibits growth of several liver cancer cell lines in vitro. Transfection of CEBPA-51 in primary human hepatocytes upregulates CEBPA mRNA as well as markers of normal liver function and significantly increases albumin secretion. Clinical candidate MTL-CEBPA comprises the SMARTICLES® liposomal formulation of CEBPA-51 for liver delivery. 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.
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.
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.
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.
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.
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.