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diseases with saRNA therapeutics.


NOV340 liposome encapsulating nucleic acid payload achieves efficient biodistribution to erythroid progenitor cells

saRNA can be delivered to therapeutically relevant ErP cells in vivo using a clinically established liposomal formulation. Rodent data demonstrates PD response. NHP data demonstrates delivery to greater than 60% of committed ErPs, and equivalence to monocytes in which clinical PD has been demonstrated. This delivery data provides foundations for the development of transformational RNAa therapeutics to treat beta-hemoglobinopathies. saRNA therapeutics have the potential to be a simpler treatment paradigm with lower treatment burden for patients when compared to ex vivo cell and gene therapy approaches.

Investigating Chemically-Modified Short Activating RNAs to Increase Nuclease Stability and Gene Activation

In this study, a library of chemically-modified saRNA modifications were synthesized and evaluated for their ability to activate gene expression. In addition, we investigated the nuclease stability of several of the modified saRNA duplexes. We have identified that an abasic carbon-based linker within the central region of the sense strand offers thermal destabilization, yet offers enhanced nuclease stability and RNA activation when compared to its parent duplex for 2 gene targets […]

RNA activation of CEBPA reduces proliferative capacity of acute myeloid leukemic cells in preclinical models

NOV340 liposomal nanoparticles effectively deliver fluorescently-labelled RNA to leukemic cells, both in vitro (AML cell lines) and in vivo (PDX mouse model). MTL-CEBPA transcriptionally activates CEBPA and increases C/EBP expression in both AML cell lines and MOLM14-xenograft mouse model. MTL-CEBPA improves Gilteritinib’s anti-leukemic activity and reduces leukemic cell growth in MOLM14-xenograft mouse model […]

Small-activating RNA therapy development for the inherited genetic disorder Aniridia

A lead saRNA was identified and, importantly, treatment with the lead rescued aniridia-mimicking phenotypic aspects of the PAX6 +/- line – i.e., rescuing downstream gene expression levels, cell motility, and the cell adhesion defects of the mutant cell line. Together these positive data provide a solid basis for onward development of a saRNA based treatment for aniridia and other PAX6-related disorders […]

TIMEPOINT, a Phase 1 study of MTL-CEBPA in combination with pembrolizumab, confirms the immunomodulatory effect of MTL-CEBPA in solid tumours

Here we report the findings from a biomarker pharmacodynamic analysis of paired baseline and cycle 2 tumor sample biopsies in 23 patients from the TIMEPOINT trial. Brightplex® IHC and digital pathology analyses of the samples for myeloid and T cell panels were undertaken, alongside gene expression (Nanostring I/O 360) […]

Interim results for Phase 1b dose expansion of MTL-CEBPA in combination with pembrolizumab in patients with advanced solid tumour malignancies

MTL-CEBPA in combination with pembrolizumab is safe and well tolerated, with encouraging early signs of activity in heavily pretreated patients across multiple tumour types. Treatment was associated with intratumoural changes supporting the hypothesis of immunomodulation by MTL-CEBPA and further investigation in combination with ICI is warranted […]

MTL-STING increases STING expression and potentiates efficacy of checkpoint inhibitor in murine preclinical model

cGAS-cGAMP-STING is essential for sensing foreign DNA from pathogens or self-DNA from dying cancer cells. Activation of this pathway is critical for the innate immune response to cancer and is necessary for the full efficacy of various cancer treatments including checkpoint and PARP inhibitors, radiotherapy, and CAR T-cells. Intense efforts have focused on triggering this pathway with cGAMPanalogs, which are small-molecule activators of STING. However, recent reports show that many cancer cells downregulate STING by promoter methylation. Emerging evidence suggests that the critical cells in which STING activation achieves maximal anti-cancer efficacy are myeloid cells in the tumour microenvironment (TME) […]

Open label, randomised, phase 2 study to evaluate the safety and efficacy of sorafenib with or without MTL-CEBPA, an immune-modulatory saRNA upregulating C/EBP-a, as second line treatment in advanced hepatocellular carcinoma (OUTREACH 2)

Enhancing SIRT1 Gene Expression Using Small Activating RNAs: A Novel Approach for Reversing Metabolic Syndrome

MTL-STING restores endogenous STING expression for improving efficacy of cancer therapeutics

Upregulation of C/EBPα Inhibits Suppressive Activity of Myeloid Cells and Potentiates Antitumor Response in Mice and Patients with Cancer

MTL-CEBPA Combined with Immunotherapy or RFA Enhances Immunological Anti-Tumor Response in Preclinical Models

Small activating RNAs lead the charge to turn up gene expression

Advances in oligonucleotide drug delivery

Oligonucleotides can be used to modulate gene expression via a range of processes including RNAi, target degradation by RNase H-mediated cleavage, splicing modulation, non-coding RNA inhibition, gene activation and programmed gene editing. As such, these molecules have potential therapeutic applications for myriad indications, with several oligonucleotide drugs recently gaining approval. However, despite recent technological advances, achieving efficient oligonucleotide delivery, particularly to extrahepatic tissues, remains a major translational limitation. Here, we provide an overview of oligonucleotide-based drug platforms, focusing on key approaches — including chemical modification, bioconjugation and the use of nanocarriers — which aim to address the delivery challenge.

Delivery of Oligonucleotides to the Liver with GalNAc: From Research to Registered Therapeutic Drug

Targeted delivery of oligonucleotides to liver hepatocytes using N-acetylgalactosamine (GalNAc) conjugates that bind to the asialoglycoprotein receptor has become a breakthrough approach in the therapeutic oligonucleotide field. This technology has led to the approval of givosiran for the treatment of acute hepatic porphyria, and there are another seven conjugates in registrational review or phase 3 trials and at least another 21 conjugates at earlier stages of clinical development. The review focuses on the use of this delivery system for small interfering RNAs (siRNAs) and antisense molecules that cause downregulation of target mRNA and protein. A number of other approaches such as anti-microRNAs and small activating RNAs are starting to exploit the technology, broadening the potential of this approach for therapeutic oligonucleotide intervention.

Phase Ib dose escalation and cohort expansion study of the novel myeloid differentiating agent MTL-CEBPA in combination with sorafenib in patients with advanced hepatocellular carcinoma (HCC)

MTL-CEBPA is the first small activating RNA to enter clinical trials and upregulates C/EBP-α, a master regulator of myeloid cell differentiation. We previously reported a favourable safety profile of MTL-CEBPA as a single agent in HCC (Sarker D et al, ASCO 2018). After discontinuation of MTL-CEBPA, 3 out of 5 patients (pts) treated with sorafenib off study had a complete response (CR) of 7-18 months duration; 2 pts of which demonstrated resolution of lung metastases for > 1 year. Here we provide new data on pts prospectively treated with MTL-CEBPA + sorafenib […]

MTL-CEBPA, a small activating RNA therapeutic up-regulating C/EBP-α, in patients with advanced liver cancer: a first-in-human, multi-centre, open-label, phase I trial

Purpose: Transcription factor C/EBP-α (CCAAT/enhancer-binding protein alpha) acts as a master regulator of hepatic and myeloid functions and multiple oncogenic processes. MTL-CEBPA is a first-in-class small activating RNA oligonucleotide drug which up-regulates C/EBP-α. Conclusions: MTL-CEBPA is the first saRNA in clinical trials and demonstrates an acceptable safety profile and potential synergistic efficacy with TKIs in HCC. These encouraging Phase I data validate targeting of C/EBP-α and have prompted MTL-CEBPA + sorafenib combination studies in HCC […]

Liver Activation of Hepatocellular Nuclear Factor-4a by Small Activating RNA Rescues Dyslipidemia and Improves Metabolic Profile

Non-alcoholic fatty liver disease (NAFLD) culminates in insulin resistance and metabolic syndrome. Because there are no approved pharmacological treatment agents for non-alcoholic steatohepatitis (NASH) and NAFLD, different signaling pathways are under investigation for drug development with the focus on metabolic pathways. Hepatocyte nuclear factor 4-alpha (HNF4A) is at the center of a complex transcriptional network where its disruption is directly linked to glucose and lipid metabolism. Resetting HNF4A expression in NAFLD is therefore crucial for re-establishing normal liver function […]

Investigating the biodistribution of MTL-CEBPA reveals delivery of small activating RNA into CD34+ cells and different types of immune cells in vivo

The aim of the study is to extend our knowledge on the biodistribution of MTL-CEBPA to cells of the immune system […]

Development of GalNAc-conjugated saRNA targeting HNF4A for treatment of metabolic disease

Hepatocyte nuclear factor 4 alpha (HNF4A) is a liver-enriched transcription factor and master regulator of hepatocyte function. HNF4A expression is downregulated in chronic liver disease, and the expression of HNF4A correlates with the level of liver dysfunction. We have developed a small activating RNAs (saRNAs) that upregulate the rodent HNF4A gene in primary hepatocytes in vitro and in vivo […]

First-in-human, first-in-class phase I study of MTL-CEBPA, a RNA oligonucleotide targeting the myeloid transcription factor C/EBP-α in patients with advanced hepatocellular cancer

MTL-CEBPA is a first-in-class therapy targeting the myeloid cell master regulator C/EBP-α. In HCC patients MTL-CEBPA demonstrated a good safety profile, induced altered gene expression in WBC and as well as anti-tumour activity. These encouraging phase I data validate targeting of CEBP-α and have prompted MTL-CEBPA + sorafenib combination studies in HCC […]

Targeting myeloid-derived suppressor cells and T cells: combination treatment with MTL-CEBPA and PD-1 antibody in a mouse syngeneic CT26 model

The study indicates enhanced anti-tumour activity when combining MTL-CEBPA with PD-1 antibody in the immunocompetent mouse CT26 colorectal cancer model. The combination treatment appears to result in increased penetration of TILs through modulation of immune activity in the tumour microenvironment […]

Targeted delivery of C/EBPa-saRNA by RNA aptamers shows anti-tumor effects in a mouse model of advanced PDAC

Pancreatic ductal adenocarcinoma (PDAC) is one of the most aggressive malignancies; it preferentially metastasizes to the liver and is the main cause of death from this disease. In previous studies, small activating RNA against CCAAT/enhancer-binding protein-α (C/EBPα-saRNA) demonstrated efficacy of PDAC in a local subcutaneous tumor model. In this study, we focused on the efficacy of C/EBPα-saRNA in advanced stage PDAC. For targeted delivery, we selected a new anti-transferrin receptor aptamer (TR14), which demonstrated a high binding affinity to target proteins. The TR14 aptamer was internalized with clathrin-mediated endocytosis, distributed in early endosome, late endosome, and lysosome subcellularly. To investigate its anti-tumor effects to advanced PDAC, we conjugated C/EBPα-saRNA to TR14.  […]

MTL-CEBPA, a drug candidate for hepatocellular carcinoma enhances efficacy of Sorafenib

MTL-CEBPA, a non-toxic therapeutic oligonucleotide formulated inside SMARTICLES (liposomal nanoparticles) is currently under a Phase I study for patients with advanced hepatocellular carcinoma (HCC). We have demonstrated in multiple in vivo liver disease models that MTL-CEBPA can improve liver function and inhibit HCC tumour growth. Here we show that upregulation of hepatic CEBPA expression by MTL-CEBPA enhances the effects of Sorafenib in a (Diethylnitrosamine) DEN induced cirrhotic HCC rat model by significantly reducing liver tumour volume after only two weeks of combination treatment when compared to untreated control or single agent treatment only […]

MTL‑CEBPA combined with radiofrequency ablation and immunotherapy enhances immunological anti‑tumour response in an HCC mouse model

The transcription factor CEBPA (CCAAT/enhancer-binding protein alpha) is recognised for its antiproliferative effects. MTL-CEBPA is a small activating RNA drug which upregulates gene expression of CEBPA and amongst other effects causes and immunomodulatory effect on peripheral granulocytes. Radiofrequency ablation (RFA) is standard treatment for some tumour types such as liver cancer and induces modulation of both innate and adaptive immune systems. To investigate any synergistic effect of MTL-CEBPA with RFA and immune checkpoint inhibition we initiated a reverse translation experiment, where syngeneic BNL hepatocellular carcinoma tumour cells were injected in the two opposite flanks of immunocompetent BALB/c mice (n=8 in each group) […]

Anti-inflammatory activity of MTL-CEBPA, a Small Activating RNA drug, in LPS-stimulated monocytes and humanized mice

Excessive or inappropriate inflammatory responses can cause serious and even fatal diseases. The CCAAT/enhancer-binding protein alpha (CEBPA) gene encodes C/EBPα, a transcription factor that plays a fundamental role controlling maturation of the myeloid lineage and is also expressed during the late phase of inflammatory responses when signs of inflammation are decreasing. MTL-CEBPA is a small activating RNA targeting for upregulation C/EBPα, currently being evaluated in a Phase 1b in hepatocellular carcinoma. Following dosing, subjects had reduced levels of pro-inflammatory cytokines and we therefore hypothesized that MTL-CEBPA has anti-inflammatory potential. The current study was conducted to determine the effects of C/EBPα saRNA – CEBPA-51 on inflammation in vitro and in vivo following endotoxin challenge […]

Treating Disease at the RNA Level with Oligonucleotides

As research on the structure and function of RNAs has rapidly expanded, so too has the number of oligonucleotide drugs in clinical development that regulate RNA. This article describes several examples and suggests where the field is headed.

Preliminary results of a first-in-human, first-in-class phase I study of MTL-CEBPA, a small activating RNA (saRNA) targeting the transcription factor C/EBP-α in patients with advanced liver cancer

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 Cmax 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

Gene activation of CEBPA using saRNA: preclinical studies of the first in human saRNA drug candidate for liver cancer

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.

Development and Mechanism of Small Activating RNA Targeting CEBPA, a Novel Therapeutic in Clinical Trials for Liver Cancer – Molecular Therapy

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.

saRNA-guided Ago2 targets the RITA complex to promoters to stimulate transcription – Cell Research

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.

Small activating RNA binds to the genomic target site in a seed-region-dependent manner – Nucleic Acids Research

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.

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