Development of MTL-CEPBA: Sarna Drug for Liver Disease

Send Orders for Reprints to [email protected] Journal Name, Year, Volume 1 Development of MTL-CEPBA: SaRNA drug for Liver Disease Helen L Lightfoot†a, Ryan L Setten†b,c, John J Rossib,c and Nagy A Habibd* aMiNA Therapeutics Limited, Translation & Innovation Hub, 80 Wood Lane, London, W12 0BZ, United Kingdom; bDepartment of Molecular and Cellular Biology, Beckman Research Institute of City of Hope, Duarte, CA, USA; cIrell & Manella Graduate School of Biological Sciences, Beckman Research Institute of City of Hope, Duarte, CA, USA; dDepartment of Surgery and Cancer, Imperial College London, London, UK. †authors contributed equally Abstract: Oligonucleotide drug development has revolutionised the drug discovery field allowing the notorious “undruggable” genome to become “druggable”. Of course, as with all, there are caveats to this new technology, which include restricted delivery and mode of actions – such drugs usually alter splicing and reduce RNA at the post- transcriptional level. Small activating RNA (saRNA)-mediated gene activation has opened a new potential therapeutic avenue for oligonucleotide drugs. SaRNAs promote endogenous transcription, a phenomenon known as RNA activation (RNAa), hence they function to increase gene expression levels. SaRNA based oligonucleotide therapeutics present great promise in expanding the “druggable” genome, with particular areas of interest including transcription factor activation and haploinsufficency. In this mini-review, we describe the pre-clinical development of the first saRNA drug to enter the clinic. This saRNA, referred to as MTL-CEPBA, targets the transcription factor CCAAT/enhancer-binding protein alpha (CEBPα), a tumour suppressor and critical regulator of hepatocyte function. MTL-CEPBA is presently in Phase I clinical trials for hepatocellularcarcinoma (HCC). The clinical development of MTL-CEPBA will provide the “proof of concept”, demonstrating that saRNAs can provide the basis for drugs which enhance gene expression and consequently improve disease outcome in patients. Keywords: MiNA Therapeutics, MTL-CEBPA, CEBPα, saRNA, HCC, Liver, RNA therapeutics.

INTRODUCTION Most of these candidates and drugs directly inhibit gene expression. The development of such oligonucleotide With the recent FDA approval of several oligonucleotide antisense drugs which have the ability to directly enhance antisense drugs, the RNA therapeutics field has made its gene expression also have great promise, particularly as they mark.1 After decades of setbacks associated with stability, can piggy back on the strategies (chemistry, delivery, off target effects, delivery and targeting, the fields of targeting) used in the development of the corresponding gene chemistry, RNA biology and genome sequencing have inhibition field. Several companies are developing gene revolutionised RNA therapeutics in terms of possible drug activation technologies including CRISPR/Cas9, structural compositions, mechanism of actions and target/indications. RNAs and small activating RNAs (saRNAs), with such Furthermore, the ability of the technology to utilize the same approaches6-7 complementing the more established mRNA production platform, developmental profiles (toxicology and therapeutics.8-9 In 2016, MTL-CEBPA was the first RNA pharmacokinetic) and delivery approaches when targeting a activating oligonucleotide antisense drug to enter clinical particular tissue, could help further reduce the bench to development. MTL-CEBPA is being developed by MiNA bedside timeline.2 This will likely lead to a rapid and robust Therapeutics (London) to treat liver disease among other pipeline of RNA therapeutics. The recent expansion of indications by directly activating the CCAAT/Enhancer- validated delivery approaches including the application of Binding Protein Alpha (CEBPα). This mini review will focus targeting agents for selective delivery of oligonucleotide on the research and pre-clinical development of MTL- antisense targeting drugs3, has also dramatically improved CEBPA for treatment of liver cancer. the targeting potential of this technology. For antisense targeting drugs, this is a time to celebrate. SARNAs Approved oligonucleotide antisense targeting drugs Short activating RNAs (saRNA) induce long lasting and function via a variety of mechanisms spanning mRNA exon sequence specific expression of their target gene (Table1). skipping to RNase-H induced mRNA cleavage; with all This remarkable finding was made in the mid 2000’s by targeting the mRNA.4-5 In addition, late stage clinical trials Long-Chen Li and colleagues when they observed short have also involved additional oligonucleotide antisense double stranded RNAs (dsRNA) targeting gene promoter targeting drugs which sequester microRNAs.5 sequences activated, rather than suppressed, transcription of p21WAF1/CIP1 (p21), VEGF, and E-cadherin.10 This newly *Address correspondence to this author at Department of Surgery and discovered phenomenon was named RNA activation (RNAa) Cancer, Imperial College London, London, UK.; E-mail: [email protected]. and the dsRNAs which cause it were subsequently termed

[Place holder, legend and figure will be replaced] Figure 1 – RNAa vs RNAi (a) saRNA and siRNA are similarly structured dsRNAs which differ only in their functional outcomes. (b)When delivered into cells siRNAs are loaded while in the cytoplasm into AGO1-4 and cause post transcriptional gene silencing (PTGS) of their target RNA. (c) PTGS can also occur in the nucleus when siRNA has been loaded into AGO2(x). (d) Additionally, siRNA can induce transcriptional gene silencing when siRNA is loaded into AGO1 and translocated into the nucleus (x). In contrast, saRNA must be loaded into AGO2 to be functional. (e) Once loaded, AGO2 is translocated into the nucleus with the help of import factors such as importin-8 (x). (f) In the nucleus, saRNA binds either directly to DNA or to chromatin bound RNA such as promoter associated transcripts or long non-coding RNA while AGO2 recruits RHA and CTR9 to the target promoter. AGO2 also binds TNCR6 proteins which might in turn recruit proteins involved in histone modification, mediator complex, and CCR4-NOT complex to the target gene promoter. (g) Following saRNA treatment, target promoters are more transcriptionally active and show: decreased H3K9me2, reduced acetylation of H3K9ac and H3K14ac, increased H3K4me2/3 and increased RNA pol-II occupancy.

‘ saRNAs’ to differentiate them from short interfering RNAs cytoplasmic shuttling of AGO2 appears to be, in part, (siR NA). Critically, it was found that the RNA interference dependent on importin-823 as knockdown of this importin 24 (RNAi) factor, Argonaute 2 (AGO2), was required for reduces the nuclear pool of AGO2. However, Importin-8 10 RNAa . Shortly after, Bethany Janowski et al showed that knockout does not completely exclude nuclear translocation progesterone receptor (PR) expression could also be induced of AGO2. This indicates other import factors may also by dsRNA targeting PR promoter.11 Endogenously, micro shuttle AGO2.25-26 RNA (miRNA) mir-373 target the promoters of e-cadherin and CSDC2 and induce their expression.12 Together, these Once in the nucleus, AGO2 can bind promoter associated transcripts27-28 that contain a complementary sequence or reports laid the groundwork for understanding how RNAa RNAs that are transcribed through a promoter region with influences gene regulation and pointed to the potential for complementary sequence (Fig1).10,29 saRNAs are also saRNA mediated gain-of-function therapy in the clinic.13,14 capable of inducing RNAa of genes when designed to target saRNAs are structurally identical to siRNA. The critical RNA transcribed from outside a gene promoter such as long design difference between the two is their intended targets. non-coding RNAs (lncRNA) (Fig1). 7, 30 Here, the targeted saRNAs act in the nucleus and are typically designed to nascent RNA likely acts as a ‘tether’ for the saRNA-AGO2 contain sequences complimentary to regions near or within complex keeping the complex in close proximity to the gene promoters10-11 while siRNAs are complimentary to target gene promoter and allowing physical contact between mRNA (Fig1).8 Both are double stranded, ~21-mer, RNA the two (Fig1). Supporting an RNA targeting model, oligonucleotides. Short dsRNAs that are introduced into a knockdown of these RNAs by antisense oligonucleotides cell are recognized in the cytoplasm by dsRNA loading (ASOs) abolishes the effect of saRNA (Fig1).29 In addition factors and subsequently loaded into one of the four AGO to saRNA targeting RNA, there is chromatin proteins (Fig1).15 The guide strand (complementary to the immunoprecipitation (ChIP) based evidence that biotinylated target RNA of interest) is retained upon loading while the saRNA might also recognize and bind directly to DNA. 13, 31 passenger strand (matching the target RNA sequence) is AGO2 is believed to recruit proteins that establish discarded. Critically, loading of AGO2 is required for 10 10-11 decreased H3K9me2 , reduced acetylation of H3K9ac and RNAa. RNA-AGO complexes have post-transcriptional 11 10-11 gene silencing (PTGS) potential in agreement with their H3K14ac , increased H3K4me2/3 and increased RNA pol-II occupancy31-33 when localized at a target gene by a canonical role in the RNA induced silencing complex saRNA (Fig.1). Still, the current mechanistic explanation for (RISC).15-16 In addition to PTGS17,18, AGO2 can induce gene specific transcriptional activation10-11 or suppression19 when RNAa remains incomplete. Complicating this, timing poses a major challenge should recruitment of any factor be a present in the nucleus.20 Nuclear AGO2 is also involved in transient process. Observable effects of RNAa on gene DNA repair21 and alternative-splicing.22 AGO proteins do not contain a nuclear localization sequence (NLS). Nuclear- expression can take anywhere between 24 to 48 hours, depending on cell line, and last up to 2 weeks.10-11, 33 This is Short Running Title of the Article Journal Name, 2017, Vol. 0, No. 0 3 in contrast to cytoplasmic and nuclear RNAi PTGS which is recognizes the sequence (G/A)TTGCG(T/C)AA(T/C) or observed in under 24 hours.18 The lag time of RNAa broadly the promoter CCAAT box sequence44, and a C- compared to RNAi might hint at a greater time requirement terminal bZIP domain containing a conserved leucine-rich for RNAa based epigenetic changes to be established at gene dimerization domain.45-47 The formation of homo- or hetero- promoters. dimers between CEBP family members and bZIP extended 3948 The question remains as to whether RNAa mediated family members is a requirement for DNA binding. protein recruitment proceeds through direct or indirect CEBPα is an intronless gene located on chromosome binding to AGO2. Using mass-spectrometry and immuno- 19q13.1. It encodes the protein CEBPα. It is transcribed from blotting, a recent study identified TNCR6A/B/C proteins and the reverse strand.49 The resulting transcript contains a AGO3 as direct binding partners of nuclear AGO2.34 A primary and alternative translation initiation codon and is follow-up study performed in a similar manner provided translated into one of two isoforms, 42 kDa (p42)* or 30 kDa further evidence that nuclear TNCR6A is tightly associated (p30).50-52 In some instances the two isoforms exhibit similar with AGO2 and is also associated with proteins involved in: biological effects. For example, both isoforms can act as histone modification, mediator complex, and CCR4-NOT transactivators of the CEBPα and adipocytes protein 2 (aP2) 35 complex (Fig1). Mass-spectrometry and immuno-blotting promoters.51 Additionally, both isoforms have inhibitory of biotinylated guide saRNA isolated from nuclei, identified potential through a negative regulatory domain found N- AGO2, RNA Helicase A (RHA), and CTR9 as proteins terminal to TAD3.50-51, 53 However, functional differences 31 associated with saRNAs (Fig1). AGO2 was shown to between the two isoforms exist. CEBPα p42 is generally directly associate with RHA and CTR9 in the presence of the more abundant52, is more often associated with saRNA.31 Efficient knockdown of RHA or CTR9 were 31 13 transactivation of target genes owing to its three TADs shown to inhibit RNAa of p21 and CEBPA . Since CTR9 (TAD1-3)50-52, and possesses anti-mitotic activity due in part is part of the polymerase associated factor complex (PAF1) - 36 to N-terminal binding of retinoblastoma (Rb) and which has a general role in transcription ; the exact role of p21.(Cip1).54-56 In contrast, CEBPα p30 does not possess anti- CTR9 with saRNA/RNAa has yet to be fully defined. mitotic activity and lacks TAD1-2.50-51 Consequently, Increased occupancy of gene promoters with transcription- CEBPα p30 cannot bind with TATA box-binding protein associated machinery could be a result of their recruitment in (TBP)43, TFIIB43, or the histone acetyltransferase CREB- an saRNA mediated fashion or a downstream consequence binding Protein (CBP)57 resulting in severely reduced of a promoter that has been made transcriptionally active by transactivation potential compared to CEBPα p42.50-51 In RNAa. While this work is an exciting advance in our some situations the isoforms can have opposite effects; in understanding of RNAa (Fig.1), further studies will be the case of G-CSF receptor expression CEBPα p30 is needed to confirm the possible roles for TNCR6A associated 58 59 proteins and other factors beyond RHA and CTR9 in the inhibitory while CEBPα p42 promotes expression. Due to RNAa process. these differences -when mutation of one allele results in defective CEBPα p42 protein - CEBPα p30 can act in a Gene Protein Reported dominant-negative fashion.60-61 CDKN1A p21WAF1/CIP1 2006 10 CEBPα is expressed across a wide range of human solid VEGFA Vascular endothelial growth 2006 10 tissues and blood cells albeit at differing levels. The solid factor A (VEGFA) tissues with highest expression based on mRNA-seq are CDH1 E-cadherin 2006 10 liver, skin, adipose, and breast followed by small intestine 62 PGR Progesterone Receptor (PR) 2007 11 and lung. Multiple regulatory mechanisms have evolved to maintain tight control over CEBPα expression in tissues at -place the transcriptional, post-transcriptional, and post- holder- translational levels. At the transcriptional level, CEBPα is -place regulated by two promoters - a core promoter (-437 to +4bp holder- from the transcriptional start site (TSS)) and distal promoter (-1400 to -600bp from the TSS) - both of which lie within a CpG island.63-64 Regardless of high or low expression, the core CEBPα promoter is generally free of DNA CEBPA CCAAT/enhancer binding 65 protein alpha (CEBPA) methylation potentially providing a platform for temporary transcription factor binding depending on stimuli. In Table 1 – Genes successfully upregulated by saRNA contrast, distal promoter methylation is inversely correlated treatment. with CEBPα expression keeping expression repressed in cell CCAAT/ENHANCER-BINDING PROTEIN ALPHA lines that do not continuously express CEBPα.65 Enhancers (CEBPΑ) have profound impact on development and cell type specific regulation of proteins.66 Enhancer-promoter associations CCAAT enhancer binding protein family members have been described in both general67 and cell type specific (CEBPs) are a family of six transcription factors (α - ζ) that CEBPα regulation.67-68 LncRNAs69 transcribed from the regulate genes involved in: cellular differentiation, 37-40 CEBPα locus have also been shown to have regulatory effect proliferation, metabolism, and immunity (Table2). on CEBPα. The anti-sense CEBPα transcript ‘adipogenic CEBPs are a sub-group of the basic region leucine zipper differentiation induced noncoding RNA’ (ADINR) was transcription factors (bZIPs) family. CEBPs are recently shown to stimulate CEBPα expression in cis during characterized as having a N-terminal transactivation adipogenesis in human mesenchymal stem cells by directly domain(s) (TAD)41-43, a DNA binding domain which binding PA1 which in turn recruits MLL3/4 histone methyl- 4 Journal Name, 2017, Vol. 0, No. 0 Principal Author Last Name et al. transferase complexes. 70 Another lncRNA, extra-coding In the field of oncology, CEBPα is known as a tumour CEBPα (ecCEBPα), is transcribed upstream and through the suppressor gene. Established hepatocyte cultures derived gene body of CEBPα and has been proposed to block DNA from CEBPα -/- mice exhibit rapid growth, accumulation of methyltransferase 1 (DNMT1) from performing maintenance chromosomal abnormalities, and form nodules when placed methylation on the CEBPα promoter.71 into abdominal subcutaneous tissue of nude mice.97 Mutations in either the TADs or bZIP domains (common in Post transcriptionally and post translationally, the leukemias) which diminish or abolish CEBPα function along turnover of CEBPα mRNA and protein in cultured cells is with general repression of CEBPα expression can promote rapid.72-73 For mRNA, a 70-80% reduction was observed tumorigenesis and tumor progression.61-62, 78-80 Increases in after 2 hours of actinomycin D treatment in hepatocytes.72 oncogenic miRNAs, such as miR-182 which silences CEBPα protein half-life was found to be 100 minutes, as CEBPα mRNA,74 and down regulation of CEBPα due to measured by pulse chase in HL-60 cells.73 MicroRNA distal promoter hyper-methylation are common in many (miRNA) mediated degradation could partially be cancers29, 98-100 including hepatocellular carcinoma.64 In responsible - miR-182 targets the 3’ UTR of CEBPα mRNA general, solid tumors exhibit deficient CEBPα expression resulting in lower CEBPα expression.74 Additional modes of rather than loss of function mutations62. Thus, targeted CEBPα regulation involve RNA binding proteins (e.g. restoration of CEBPα in HCC holds clinical promise. calreticulin (CRT)) which bind stem-loop structures within the CEBPα transcript inhibiting translation75 and post- Beyond HCC, functional inhibition of CEBPα in translational modifications which effect CEBPα stability hepatocytes might be partially responsible for lipid (e.g. phosphorylation by c-Jun N-terminal kinase (JNK) 1)73, accumulation observed in NASH. PPARγ and CEBPα 76-77. Unsurprisingly, misregulation of CEBPα by defects in promote lipolysis and decrease triglyceride content in fully these regulatory mechanisms correlates with disease states in differentiated adipocytes.101 Finally, given the role for afflicted tissues. 61-62, 64, 78-80 CEBPα in gluconeogensis81 in the liver and in adipogenesis102 it will be of interest to determine what CEPBA IN LIVER FUNCTION effects, if any, CEBPα function has on resting glucose levels CEBPα `s high abundance in the liver suggests a critical and insulin resistance with regard to type II diabetes. Taken role in liver function. Indeed, CEBPα -/- mouse neonates fail together, restoration of CEBPα function in liver disease has to store hepatic glycogen and die rapidly (within 8 hours) powerful therapeutic potential. after birth due to hypoglycemia.81 Livers from these CEBPα -/- Regulation Gene Function neonates displayed reduced albumin, glycogen synthase, 81 PEPCK, GLUT2, and G6Pase mRNA levels compared to + ABL plasma colloid osmotic pressure, WT or heterozygous mice (Table2). In addition, CEBPα -/- carrier protein mice develop hyperammonemia resulting from impaired - expression of ornithine cycle enzymes.82 These findings along with the observations of anti-mitotic effects54-56 of CEBPα, confirm a major role for CEBPα in liver physiology.

CEPBA IN LIVER DISEASE AND LIVER CANCER Common liver diseases include: non-alcoholic steatohepatitis (NASH) or Fatty Liver Disease, alcohol associated steatohepatitis (ASH), and hepatitis B/C; all of Table 2 – A non-exhaustive list of genes regulated by which increased risk of impaired liver function.83-86 If left CEBPA. (+) CEBPA activates gene expression. (-) CEBPA untreated, such diseases can lead to further problems represses gene expression. resulting from consequent cirrhosis of the liver and liver cancer.83-88 Liver cancer is the second most common cause of DEVELOPMENT OF CEBPΑ SARNA FOR LIVER cancer-associated death worldwide in men and the sixth in DISEASE women where HCC accounts for 70-80% of all cases.89-90 Surgical resection of tumors is the preferred method of treatment. While such resection has the potential to cure saRNA design patients and improve long-term survival compared to other CEBPα saRNAs were designed against the CEBPα methods, this option is only possible in about 5-15 % of promoter region as described by Voutila et al.103 CEBPα cases.91 Poor liver function or existence of tumors that have saRNA selection considers factors such as the removal of invaded into the surrounding vasculature can prohibit polymeric motifs, Guanine-Cytosine (GC) content, off target therapeutics options which would further compromise the effects, target composition and predicted saRNA activity. liver.92 Consequently, barring a liver transplant, prognosis for patients is generally poor with an overall 5-year survival Proof of concept studies rate ranging from 31% in early disease to 3% in metastatic CEBPα saRNA transfection into a human HCC cells line, 93 94 disease. Currently, sorafenib - a multikinase inhibitor of HepG2 increased CEBPα mRNA in a dose-dependent Raf-1, B-Raf, VEGFR, and PDGFR - represents the manner, and consequently induced albumin mRNA standard-of-care treatment for advanced HCC. However, expression and protein secretion, consistent with CEBPα`s treatment only prolongs the median life expectancy by 3 role in liver function.7 A reduction in methylation at CpG 95-96 months. Therefore, there is still significant unmet islands in both CEBPα and albumin promoter regions was medical need in the treatment of HCC. observed, where CEBPα binding motifs were also present. Short Running Title of the Article Journal Name, 2017, Vol. 0, No. 0 5

The expression of other downstream markers (i.e. ornithine To further develop CEPBA-51 as a clinical candidate, the transcarbamylase (OTC) and Alpha-fetoprotein (AFP)) were effect of chemical modifications on activity and immune also altered as expected due to their role in hepatocyte stimulation in primary human PMBCs was assessed. 2`-O- maintenance and liver differentiation. Microarray analysis of methyl modifications on the sense strand were well tolerated, 84 liver specific genes, identified additional factors altered and upon treatment with one compound limited TNF-α and upon CEBPα saRNA treatment. Down-regulated genes were IFN-α secretion was observed. This suggested particular 2`- strongly enriched in functions related to negative regulation O-methyl modification patterns within saRNAs display of apoptosis and cell death, whereas up-regulated genes were limited immune activation. Furthermore, addition of a enriched in functions related to positive regulation of cell 5`inverted abasic modification105 on the sense strand further differentiation. For c-Myc and STAT3, an increase in the enhanced activity, through directing Ago2 loading towards methylation state of their promoters was suggested to be the functional antisense strand.13 Based on these promising responsible for their reduced expression; again, CEBPα observations, the chemically improved CEPBA-51 saRNA binding motifs were located in their promoter regions. was taken forward into pre-clinical trials for liver disease. Phenotypically, CEBPα saRNA reduced proliferation (50 %) of HepG2 cells in a dose-dependent manner.7 This study Mechanistic Investigations demonstrated that a saRNA targeting the CEBPα promoter can increase CEBPα levels and consequently improve Investigations into the mechanism of action of CEBPα surrogate markers of liver function in a HCC cell system. saRNAs, utilizing a variety of approaches (reporter, nuclear run-on, mutagenesis/chemical modification, Argonaute Intravenous delivery of the CEBPα saRNA complexed 2 (Ago2) knock out and Ago1-4 pull down studies) in with polyamidoamine (PAMAM) dendrimer, a nanoparticle predominantly HepG2 cells confirmed CEBPα saRNA which preferentially accumulate in PBMCs and liver104 into functions at the transcriptional level, with such activity diethylnitrosamine (DEN)-induced rat HCC model, selectively associated with the CEBPα promoter.13 saRNA upregulated CEBPα and albumin mRNA, as well as altered activity was Ago2 dependant, with a requirement for saRNA expression of critical hepatocyte markers such as HNF1α and guide strand loading but not Ago2 induced RNA cleavage.13 HNF4α in liver tissue.7 Furthermore, significant increases in Although still not understood, the level of the antisense RNA serum albumin and significant decreases in serum bilirubin, was upregulated. Paf1/RNA polymerase II complex aspartate aminotransferase (AST) and alanine component (CTR9), a factor previously found to be involved aminotransferase (ALT) were also observed. Histological with the transcriptional driven RNAa mechanism31 was examination and immunohistology studies of the liver required for CEBPα saRNA activity - further confirming the showed a CEBPα saRNA induced reduction in tumour mechanism of CEBPα saRNA was distinct from that of burden (80 %) and pre(neoplasmtic) marker GST-P levels siRNAs.13 Interestingly, the saRNA CEBPA-51 was capable (40 %).7 This exciting study confirmed that CEBPα saRNA of activating CEBPA expression in HepG2 within 72 hours is functional in vivo and provided the basis to further develop of transfection despite the upstream promoter remaining CEBPα saRNA for clinical use. methylated.7

SaRNA optimisation Pre-clinical and clinical Prior to pre-clinical studies, the CEBPα saRNA For efficient delivery, CEBPA-51 is encapsulated in compound was optimised with regards to its composition and Marina Biotech’s liposomal carrier molecules target CEBPα sequence. Bioinformatic analysis of the (SMARTICLES) and is referred to as MTL-CEBPA. CEBPα promoter locus identified two hot spots for saRNA SMARTICLES are amphoteric liposomes with anionic and activity.13 Such hot spots were located in the coding region cationic groups that provide serum stability and high tropism of CEBPα, where a non-coding RNA overlaps in the to the liver with efficient cellular uptake. Furthermore, they antisense orientation. Nucleotide walk across these two provide a pH-triggered endosomal escape for intracellular hotspots produced several candidates whose activity were delivery of the double-stranded saRNA payload. then assessed in HepG2 cells. The candidate with the Delivery of MTL-CEBPA-51 into a diethylnitrosamine strongest potency was selected (CEBPα-51; 2.5-fold) and a (DEN)-induced cirrhotic HCC rat model (refs), displayed dose response observed with an EC50 of 5.36 nM. This similar results as described in Reebye et al.7 increased saRNAs target site was located at the same hot spot that of 7 CEPBA mRNA expression, an 80 % reduction in tumour the original saRNA described above (CEPBA-AVI ). size and a decrease in relevant liver parameters (i.e. AST, Corresponding increases in CEBPα protein were observed as ALT) 1 week after treatment, compared to the negative well as an increase in the downstream CEBPα marker 7 control oligonucleotide (refs). Additional studies in CCl4- albunium mRNA. As shown for CEPBA-AVI , induced liver failure rat models showed significant phenotypically, CEBPA-51 reduced proliferation of HepG2 improvements in clinically relevant parameters, including an 13 and Hep3B cells. Off target effects (siRNA and miRNA) increase in serum albumin and improvements in additional were assessed in a variety of species. The target site for markers for liver function (i.e. AST, ALT and CEPBA-51 was highly conserved (human, non-human Hydroxyrproline) upon treatment with MTL-CEBPA-51. primate and rodent). Furthermore, CEBPα saRNA activity Furthermore, liver toxicity and function, as well as overall was also conserved across species. Although one or two survival were significantly improved following MTL- mismatched targets were identified, no significant effects on CEBPA-51 treatment (refs). Further in vivo studies involving six relevant liver or cancer biology targets were observed methionine choline deficient diet induced non-alcoholic 13 upon CEBPα saRNA treatment. steatohepatitis mice, Thioacetamide (TAA) induced acute liver failure rats and a nude mouse liver orthotopic xenograft 6 Journal Name, 2017, Vol. 0, No. 0 Principal Author Last Name et al. tumour mice (refs) demonstrated that in a diverse range of 6. Cyranoski, D., CRISPR gene-editing tested in a models CEBPA-51 treatment improves a variety of different person for the first time. Nature 2016, 539 (7630), 479. liver parameters. Furthermore, liver tumour formation, and 7. Reebye, V.; Saetrom, P.; Mintz, P. J.; Huang, K. intrahepatic and distant lung tumour formation were lower in W.; Swiderski, P.; Peng, L.; Liu, C.; Liu, X.; Lindkaer- CEBPA-51 treated mice.106 Combined, this data strongly Jensen, S.; Zacharoulis, D.; Kostomitsopoulos, N.; Kasahara, supported clinical exploration of CEBPA-51 in treatment of N.; Nicholls, J. P.; Jiao, L. R.; Pai, M.; Spalding, D. R.; liver fibrosis and other liver disease. MTL-CEBPA-51 is Mizandari, M.; Chikovani, T.; Emara, M. M.; Haoudi, A.; presently being assessed in a Phase I clinical trial for patients Tomalia, D. A.; Rossi, J. J.; Habib, N. A., Novel RNA with advanced liver cancer. This clinical study represents the oligonucleotide improves liver function and inhibits liver first-in-human trial of an activating small RNA carcinogenesis in vivo. Hepatology 2014, 59 (1), 216-27. oligonucleotide therapy. 8. Itaka, K., Development of mRNA-based CONCLUSION therapeutics. Nihon Yakurigaku Zasshi 2016, 148 (4), 190- 196. The mono-therapeutic one drug - one target nature of 9. Sergeeva, O. V.; Koteliansky, V. E.; Zatsepin, T. S., antisense functioning drugs has created an obstacle for their use in complex diseases where the pathogenic state is made mRNA-Based Therapeutics - Advances and Perspectives. up of multiple dysregulated redundant pathways (i.e. cancer). Biochemistry (Mosc) 2016, 81 (7), 709-22. Basically, inhibition of a single gene does not have an effect. 10. Li, L. C.; Okino, S. T.; Zhao, H.; Pookot, D.; Place, Targeting transcription factors, with their multiple R. F.; Urakami, S.; Enokida, H.; Dahiya, R., Small dsRNAs connections linking redundant pathways acting in union to induce transcriptional activation in human cells. Proc Natl drive disease state, allows such drugs to function as wide Acad Sci U S A 2006, 103 (46), 17337-42. spread pathway regulators - still however targeting a single 11. Janowski, B. A.; Younger, S. T.; Hardy, D. B.; gene. saRNAs are part of a restricted repertoire of drugs Ram, R.; Huffman, K. E.; Corey, D. R., Activating gene which can selectively enhance transcription factor expression in mammalian cells with promoter-targeted expression, modulating their extensive yet directed network. duplex RNAs. Nat Chem Biol 2007, 3 (3), 166-73. CEBPα is an attractive transcription factor target for saRNA 12. Place, R. F.; Li, L. C.; Pookot, D.; Noonan, E. J.; development in advanced HCC, which at present has limited Dahiya, R., MicroRNA-373 induces expression of genes treatment options due to poor liver function or large with complementary promoter sequences. Proc Natl Acad unresectable tumours making them ineligible for surgical Sci U S A 2008, 105 (5), 1608-13. intervention. CEBPα has proven anticancer properties, strong 13. Voutila, J.; Reebye, V.; Roberts, T. C.; Protopapa, association with disease severity in patients, and is critical P.; Andrikakou, P.; Blakey, D. C.; Habib, R.; Huber, H.; for hepatocyte function. The findings that an saRNA Saetrom, P.; Rossi, J. J.; Habib, N. A., Development and targeting CEBPα reduce tumour burden and improve liver Mechanism of Small Activating RNA Targeting CEBPA, a function in animal models provided the basis for it`s Novel Therapeutic in Clinical Trials for Liver Cancer. Mol advancement into the clinic. We hope that by improving Ther 2017, 25 (12), 2705-2714. liver function, saRNAs targeting CEBPα will improve the 14. Sarker, D.; Plummer, E. R.; Basu, B.; Meyer, T.; outcomes for patients which are ineligible for surgical Huang, K.-W.; Evans, T. R. J.; Spalding, D.; Ma, Y. T.; intervention, as well as induce liver regeneration in a range of additional liver-related indications. Palmer, D. H.; Chee, C. E.; Toh, H. C.; Habib, N. 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