The Effect of Interleukin-10 Immunotherapy on Renal Ischemia-Reperfusion Injury: A Systematic Review and Meta-Analysis of Preclinical Studies.

Renal ischemia-reperfusion is a common cause of acute kidney injury leading to significant morbidity and mortality. There are no effective treatments available in clinical practice. This meta-analysis aims to assess the effect of IL-10 immunotherapy on renal ischemia-reperfusion injury. Medline, Embase, Cochrane-library, Google Scholar and clinicaltrials.gov were searched up to 31 March 2023. Preclinical and clinical interventional studies investigating IL-10 immunotherapy for renal ischemia-reperfusion were eligible for inclusion. The primary endpoint was renal function (serum creatinine) following ischemia-reperfusion. The secondary endpoints included mitochondrial integrity, cellular proliferation, regulated cell death (TUNEL assay), expression of inflammatory cytokines (TNF-α, IL-6 and IL-1ß), M1/M2 macrophage polarization, tissue integrity (tubular injury score), long-term kidney fibrosis (fibrotic area %) and adverse events (pulmonary toxicity, cardiotoxicity hepatotoxicity). The search returned 861 records. From these, 16 full texts were screened and subsequently, seven animal studies, corresponding to a population of 268 mice/rats, were included. Compared to the control treatment, IL-10 immunotherapy reduced serum creatinine more effectively within 24 h of administration (95% CI: -9.177, -5.601, I2 = 22.42%). IL-10 immunotherapy promoted mitochondrial integrity and cellular proliferation and reduced regulated cell death (95% CI: -11.000, -4.184, I2 = 74.94%). It decreased the expression of TNF-α, IL-6 and IL-1ß, led to M2 polarization of the local macrophages, reduced tubular injury score (95% CI: -8.917, -5.755, I2 = 22.71%), and long-term kidney fibrosis (95% CI: -6.963, -3.438, I2 = 0%). No adverse outcomes were captured. In Conclusion, IL-10 immunotherapy safely improves outcomes in animal models of renal ischemia-reperfusion; the translational potential of IL-10 immunotherapy needs to be further investigated in clinical trials.

Cannabidiol's cytotoxicity in pancreatic cancer is induced via an upregulation of ceramide synthase 1 and ER stress.

Pancreatic ductal adenocarcinoma (PDAC) remains one of the most aggressive malignancies with a median 5 year-survival rate of 12%. Cannabidiol (CBD) has been found to exhibit antineoplastic potential and may potentiate the anticancer effects of cytotoxic's such as gemcitabine. CBD therapy has been linked to de novo synthesis of ceramide. The sphingolipid ceramide is a potent tumour suppressor lipid with roles in apoptosis and autophagy. One of the key players involved is ceramide synthase, an enzyme with six isoforms (CerS1-CerS6), reported to have disease prognostic value. Quantitative real time PCR was used to determine mRNA expression levels of ceramide synthase isoforms, GRP78, ATF4 and CHOP. Western blotting was used to analyze protein expression of these markers and knockdown of CerS1 and GRP78 were applied via an siRNA and confirmed by the two mentioned methods. Mice with PDAC xenografts were injected via intraperitoneal method with drugs and tumours were analysed with flow cytometry and processed using H&E and IHC staining. siRNA knockdown of ceramide synthase 1 (CerS1) and analysis point to evidence of a putative CerS1 dependent pathway driven by CBD in activating endoplasmic reticulum (ER) stress target; GRP78. Upon CBD treatment, CerS1 was upregulated and downstream this led to the GRP78/ATF4/CHOP arm of the unfolded protein response (UPR) pathway being activated. In an in vivo model of PDAC in which CerS1 was not upregulated on IHC, there was no observed improvement in survival of animals, however a reduction in tumour growth was observed in combination chemotherapy and CBD group, indicating further investigations in vivo. These findings provide evidence of a potential ceramide induced cytotoxic mechanism of action of CBD in pancreatic ductal adenocarcinoma.

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

Metabolic syndrome (MetS) is a pathological condition characterized by abdominal obesity, insulin resistance, hypertension, and hyperlipidemia. Sirtuin 1 (SIRT1), a highly conserved histone deacetylase, is characterized as a key metabolic regulator and protector against aging-associated pathologies, including MetS. In this study, we investigate the therapeutic potential of activating SIRT1 using small activating RNAs (saRNA), thereby reducing inflammatory-like responses and re-establishing normal lipid metabolism. SIRT1 saRNA significantly increased SIRT1 messenger RNA (mRNA) and protein levels in both lipopolysaccharide-stimulated and nonstimulated macrophages. SIRT1 saRNA significantly decreased inflammatory-like responses, by reducing mRNA levels of key inflammatory cytokines, such as Tumor Necrosis Factor alpha, Interleukin 1 beta (IL-1ß), Interleukin 6 (IL-6), and chemokines Monocyte Chemoattractant Protein-1 and keratinocyte chemoattractant. SIRT1 overexpression also significantly reduced phosphorylation of nuclear factor-κB and c-Jun N-terminal kinase, both key signaling molecules for the inflammatory pathway. To investigate the therapeutic effect of SIRT1 upregulation, we treated a high-fat diet model with SIRT1 saRNA conjugated to a transferrin receptor aptamer for delivery to the liver and cellular internalization. Animals in the SIRT1 saRNA treatment arm demonstrated significantly decreased weight gain with a significant reduction in white adipose tissue, triglycerides, fasting glucose levels, and intracellular lipid accumulation. These suggest treatment-induced changes to lipid and glucose metabolism in the animals. The results of this study demonstrate that targeted activation of SIRT1 by saRNAs is a potential strategy to reverse MetS.

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

The transcription factor CEBPA is a master regulator of liver homeostasis, myeloid cell differentiation and is downregulated in several oncogenic diseases. MTL-CEBPA is a small activating RNA drug which upregulates gene expression of CEBPA for treatment of hepatocellular carcinoma (HCC). We investigate whether MTL-CEBPA has immune modulatory effects by combining MTL-CEBPA with an anti-PD-1 checkpoint inhibitor (CPI) and/or radiofrequency ablation (RFA) in two preclinical models. First, mice with two flanks of HCC tumors (BNL) were treated with combinations of RFA (right flank), anti-PD-1 or MTL-CEBPA. The reduction of the left flank tumors was most pronounced in the group treated with RFA+anti-PD1+MTL-CEBPA and 7/8 animals responded. This was the only group with a significant increase in CD8+ and CD49b+/CD45+ tumor infiltrating lymphocytes (TIL). Second, a combination of anti-PD-1+MTL-CEBPA was tested in a CT26 colon cancer model and this treatment significantly reduced tumor size, modulated the tumor immune microenvironment and increased TILs. These data suggest a clinical role for combination treatment with CPIs, RFA and MTL-CEBPA through synergistic priming of the immune tumor response, enabling RFA and CPIs to have a pronounced anti-tumor effect including activity in non-treated tumors in the case of RFA.

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

PURPOSE: To evaluate the mechanisms of how therapeutic upregulation of the transcription factor, CCAAT/enhancer-binding protein alpha (C/EBPα), prevents tumor progression in patients with advanced hepatocellular carcinoma (HCC) and in different mouse tumor models. EXPERIMENTAL DESIGN: We conducted a phase I trial in 36 patients with HCC (NCT02716012) who received sorafenib as part of their standard care, and were given therapeutic C/EBPα small activating RNA (saRNA; MTL-CEBPA) as either neoadjuvant or adjuvant treatment. In the preclinical setting, the effects of MTL-CEBPA were assessed in several mouse models, including BNL-1ME liver cancer, Lewis lung carcinoma (LLC), and colon adenocarcinoma (MC38). RESULTS: MTL-CEBPA treatment caused radiologic regression of tumors in 26.7% of HCC patients with an underlying viral etiology with 3 complete responders. MTL-CEBPA treatment in those patients caused a marked decrease in peripheral blood monocytic myeloid-derived suppressor cell (M-MDSC) numbers and an overall reduction in the numbers of protumoral M2 tumor-associated macrophages (TAM). Gene and protein analysis of patient leukocytes following treatment showed CEBPA activation affected regulation of factors involved in immune-suppressive activity. To corroborate this observation, treatment of all the mouse tumor models with MTL-CEBPA led to a reversal in the suppressive activity of M-MDSCs and TAMs, but not polymorphonuclear MDSCs (PMN-MDSC). The antitumor effects of MTL-CEBPA in these tumor models showed dependency on T cells. This was accentuated when MTL-CEBPA was combined with checkpoint inhibitors or with PMN-MDSC-targeted immunotherapy. CONCLUSIONS: This report demonstrates that therapeutic upregulation of the transcription factor C/EBPα causes inactivation of immune-suppressive myeloid cells with potent antitumor responses across different tumor models and in cancer patients. MTL-CEBPA is currently being investigated in combination with pembrolizumab in a phase I/Ib multicenter clinical study (NCT04105335).

Cannabinoids in the landscape of cancer.

INTRODUCTION: Cannabinoids are a group of terpenophenolic compounds derived from the Cannabis sativa L. plant. There is a growing body of evidence from cell culture and animal studies in support of cannabinoids possessing anticancer properties. METHOD: A database search of peer reviewed articles published in English as full texts between January 1970 and April 2021 in Google Scholar, MEDLINE, PubMed and Web of Science was undertaken. References of relevant literature were searched to identify additional studies to construct a narrative literature review of oncological effects of cannabinoids in pre-clinical and clinical studies in various cancer types. RESULTS: Phyto-, endogenous and synthetic cannabinoids demonstrated antitumour effects both in vitro and in vivo. However, these effects are dependent on cancer type, the concentration and preparation of the cannabinoid and the abundance of receptor targets. The mechanism of action of synthetic cannabinoids, (-)-trans-Δ9-tetrahydrocannabinol (Δ9-THC) and cannabidiol (CBD) has mainly been described via the traditional cannabinoid receptors; CB1 and CB2, but reports have also indicated evidence of activity through GPR55, TRPM8 and other ion channels including TRPA1, TRPV1 and TRPV2. CONCLUSION: Cannabinoids have shown to be efficacious both as a single agent and in combination with antineoplastic drugs. These effects have occurred through various receptors and ligands and modulation of signalling pathways involved in hallmarks of cancer pathology. There is a need for further studies to characterise its mode of action at the molecular level and to delineate efficacious dosage and route of administration in addition to synergistic regimes.

MTL-CEBPA, a Small Activating RNA Therapeutic Upregulating C/EBP-α, in Patients with Advanced Liver Cancer: A First-in-Human, Multicenter, 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 that upregulates C/EBP-α. PATIENTS AND METHODS: We conducted a phase I, open-label, dose-escalation trial of MTL-CEBPA in adults with advanced hepatocellular carcinoma (HCC) with cirrhosis, or resulting from nonalcoholic steatohepatitis or with liver metastases. Patients received intravenous MTL-CEBPA once a week for 3 weeks followed by a rest period of 1 week per treatment cycle in the dose-escalation phase (3+3 design). RESULTS: Thirty-eight participants have been treated across six dose levels (28-160 mg/m2) and three dosing schedules. Thirty-four patients were evaluable for safety endpoints at 28 days. MTL-CEBPA treatment-related adverse events were not associated with dose, and no maximum dose was reached across the three schedules evaluated. Grade 3 treatment-related adverse events occurred in nine (24%) patients. In 24 patients with HCC evaluable for efficacy, an objective tumor response was achieved in one patient [4%; partial response (PR) for over 2 years] and stable disease (SD) in 12 (50%). After discontinuation of MTL-CEBPA, seven patients were treated with tyrosine kinase inhibitors (TKIs); three patients had a complete response with one further PR and two with SD. 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-4α 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. Here, small activating RNA (saRNA) specific for upregulating HNF4A was injected into rats fed a high-fat diet for 16 weeks. Intravenous delivery was carried out using 5-(G5)-triethanolamine-core polyamidoamine (PAMAM) dendrimers. We observed a significant reduction in liver triglyceride, increased high-density lipoprotein/low-density lipoprotein (HDL/LDL) ratio, and decreased white adipose tissue/body weight ratio, all parameters to suggest that HNF4A-saRNA treatment induced a favorable metabolic profile. Proteomic analysis showed significant regulation of genes involved in sphingolipid metabolism, fatty acid ß-oxidation, ketogenesis, detoxification of reactive oxygen species, and lipid transport. We demonstrate that HNF4A activation by oligonucleotide therapy may represent a novel single agent for the treatment of NAFLD and insulin resistance.

Targeted Delivery of C/EBPα-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. Treatment of pancreatic cancer cells with the conjugates upregulated expression of C/EBPα and its downstream target p21, and inhibited cell proliferation. For in vivo assays, we established an advanced PDAC mouse model by engrafting luciferase reporter-PANC-1 cells directly into the livers of non-obese diabetic/severe combined immunodeficiency (NOD/SCID) mice. After treatment of aptamer-C/EBPα conjugates, we observed significant reduction of tumor growth in this advanced PDAC mouse model. Combinational treatment of the conjugates with gemcitabine also demonstrated enhanced anti-tumor effects in advanced PDAC. This suggests that aptamer-C/EBPα conjugates could be used as an adjuvant, along with other conventional anti-cancer drugs in advanced PDAC. In conclusion, targeted delivery of C/EBPα-saRNAs by aptamers might have potential therapeutic effects in advanced PDAC.

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 in 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, a small activating RNA targeting for upregulation of C/EBPα, is currently being evaluated in a phase 1b trial for treatment of hepatocellular carcinoma. After 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 after endotoxin challenge. CEBPA-51 led to increased expression of the C/EBPα gene and inhibition of pro-inflammatory cytokines in THP-1 monocytes previously stimulated by E. coli-derived lipopolysaccharide (LPS). Treatment with MTL-CEBPA in an LPS-challenged humanized mouse model upregulated C/EBPα mRNA, increased neutrophils, and attenuated production of several key pro-inflammatory cytokines, including TNF-α, IL-6, IL-1ß, and IFN-γ. In addition, a Luminex analysis of mouse serum revealed that MTL-CEBPA reduced pro-inflammatory cytokines and increased the anti-inflammatory cytokine IL-10. Collectively, the data support further investigation of MTL-CEBPA in acute and chronic inflammatory diseases where this mechanism has pathogenic importance.

Mechanisms involved in the activation of C/EBPα by small activating RNA in hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is generally accompanied by high mortality and low cure rate. CCAAT enhancer-binding proteins (CEBPs) are transcriptional regulators that play a key role in maintaining liver function. Altered expression of C/EBPα and C/EBPß occurs in many tumours including HCC. saRNAs are small double-stranded RNAs that enhance target gene expression at the transcriptional level. In this report, we activate CEPBA with saRNAs and suppress CEBPB with siRNAs in cells that represent three different degrees of HCC. We performed functional assays to investigate the effects of enhancing C/EBPα and its downstream targets, p21 and albumin across these lines. We also used Mass-spectrometry (MS) subsequent to a ChIP pull-down assay to characterise the components of the protein complex involved in regulating saRNA function. Putative saRNA interacting protein candidates that were identified by MS were knocked-down with siRNAs to investigate its impact on saRNA activity. We confirmed CEBPA-saRNA decreased proliferation and migration in the differentiated lines (HepG3/Hep3B). The undifferentiated line (PLCPRF5) showed saRNA-induced increase in CEBPA but with no loss in proliferation. This effect was reversed when CEBPB was suppressed with CEBPB-siRNA. When interrogating saRNA mode of action; three saRNA interacting proteins, CTR9, HnRNPA2/B1 and DDX5 were identified by MS. Targeted knock-down of these two proteins (by siRNA) abrogated saRNA activity. This study provides insight into how different HCC lines are affected by CEBPA-saRNAs and that endogenous abundance of CEBPB and saRNA accessory proteins may dictate efficacy of CEBPA-saRNA when used in a therapeutic context.

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

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.

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.

Treatment of Liver Cancer by C/EBPA saRNA.

The prognosis for hepatocellular carcinoma (HCC) remains poor and has not improved in over two decades. Most patients with advanced HCC who are not eligible for surgery have limited treatment options due to poor liver function or large, unresectable tumors. Although sorafenib is the standard-of-care treatment for these patients, only a small number respond. For the remaining, the outlook remains bleak. A better approach to target "undruggable" molecular pathways that reverse HCC is therefore urgently needed. Small activating RNAs (saRNAs) may provide a novel strategy to activate expression of genes that become dysregulated in chronic disease. The transcription factor CCAAT/enhancer-binding protein alpha (C/EBPα), a critical regulator of hepatocyte function, is suppressed in many advanced liver diseases. By using an saRNA to activate C/EBPα, we can exploit the cell's own transcription machinery to enhance gene expression without relying on exogenous vectors that have been the backbone of gene therapy. saRNAs do not integrate into the host genome and can be modified to avoid immune stimulation. In preclinical models of liver disease, treatment with C/EBPα saRNA has shown reduction in tumor volume and improvement in serum markers of essential liver function such as albumin, bilirubin, aspartate aminotransferase (AST), and alanine transaminase (ALT). This saRNA that activates C/EBPα for advanced HCC is the first saRNA therapy to have entered a human clinical trial. The hope is that this new tool will help break the dismal 20-year trend and provide a more positive prognosis for patients with severe liver disease.

Aptamer-Drug Conjugates of Active Metabolites of Nucleoside Analogs and Cytotoxic Agents Inhibit Pancreatic Tumor Cell Growth.

Aptamer-drug conjugates (ApDCs) have the potential to improve the therapeutic index of traditional chemotherapeutic agents due to their ability to deliver cytotoxic drugs specifically to cancer cells while sparing normal cells. This study reports on the conjugation of cytotoxic drugs to an aptamer previously described by our group, the pancreatic cancer RNA aptamer P19. To this end, P19 was incorporated with gemcitabine and 5-fluorouracil (5-FU), or conjugated to monomethyl auristatin E (MMAE) and derivative of maytansine 1 (DM1). The ApDCs P19-dFdCMP and P19-5FdUMP were shown to induce the phosphorylation of histone H2AX on Ser139 (γ-H2AX) and significantly inhibited cell proliferation by 51%-53% in PANC-1 and by 54%-34% in the gemcitabine-resistant pancreatic cancer cell line AsPC-1 (p ≤ 0.0001). P19-MMAE and P19-DM1 caused mitotic G2/M phase arrest and inhibited cell proliferation by up to 56% in a dose-dependent manner when compared to the control group (p ≤ 0.001). In addition, the cytotoxicity of P19-MMAE and P19-DM1 in normal cells and the control human breast cancer cell line MCF7 was minimal. These results suggest that this approach may be useful in decreasing cytotoxic side effects in non-tumoral tissue.

Targeted Delivery of C/EBPα -saRNA by Pancreatic Ductal Adenocarcinoma-specific RNA Aptamers Inhibits Tumor Growth In Vivo.

The 5-year survival rate for pancreatic ductal adenocarcinoma (PDAC) remains dismal despite current chemotherapeutic agents and inhibitors of molecular targets. As the incidence of PDAC constantly increases, more effective multidrug approaches must be made. Here, we report a novel method of delivering antitumorigenic therapy in PDAC by upregulating the transcriptional factor CCAAT/enhancer-binding protein-α (C/EBPα), recognized for its antiproliferative effects. Small activating RNA (saRNA) duplexes designed to increase C/EBPα expression were linked onto PDAC-specific 2'-Fluropyrimidine RNA aptamers (2'F-RNA) - P19 and P1 for construction of a cell type-specific delivery vehicle. Both P19- and P1-C/EBPα-saRNA conjugates increased expression of C/EBPα and significantly suppressed cell proliferation. Tail vein injection of the saRNA/aptamer conjugates in PANC-1 and in gemcitabine-resistant AsPC-1 mouse-xenografts led to reduced tumor size with no observed toxicity. To exploit the specificity of the P19/P1 aptamers for PDAC cells, we also assessed if conjugation with Cy3 would allow it to be used as a diagnostic tool on archival human pancreatic duodenectomy tissue sections. Scoring pattern from 72 patients suggested a positive correlation between high fluorescent signal in the high mortality patient groups. We propose a novel aptamer-based strategy for delivery of targeted molecular therapy in advanced PDAC where current modalities fail.

Novel RNA oligonucleotide improves liver function and inhibits liver carcinogenesis in vivo.

UNLABELLED: 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.

Exploiting human CD34+ stem cell-conditioned medium for tissue repair.

Despite the progress in our understanding of genes essential for stem cell regulation and development, little is known about the factors secreted by stem cells and their effect on tissue regeneration. In particular, the factors secreted by human CD34+ cells remain to be elucidated. We have approached this challenge by performing a cytokine/growth factor microarray analysis of secreted soluble factors in medium conditioned by adherent human CD34+ cells. Thirty-two abundantly secreted factors have been identified, all of which are associated with cell proliferation, survival, tissue repair, and wound healing. The cultured CD34+ cells expressed known stem cell genes such as Nanog, Oct4, Sox2, c-kit, and HoxB4. The conditioned medium containing the secreted factors prevented cell death in liver cells exposed to liver toxin in vitro via inhibition of the caspase-3 signaling pathway. More importantly, in vivo studies using animal models of liver damage demonstrated that injection of the conditioned medium could repair damaged liver tissue (significant reduction in the necroinflammatory activity), as well as enable the animals to survive. Thus, we demonstrate that medium conditioned by human CD34+ cells has the potential for therapeutic repair of damaged tissue in vivo.

A Short-activating RNA Oligonucleotide Targeting the Islet ß-cell Transcriptional Factor MafA in CD34(+) Cells.

Upon functional loss of insulin producing islet ß-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 (2013) 2, e97; doi:10.1038/mtna.2013.23; advance online publication 4 June 2013.

Role of the HSP90-associated cochaperone p23 in enhancing activity of the androgen receptor and significance for prostate cancer.

Prostate tumor growth initially depends on androgens, which act via the androgen receptor (AR). Despite androgen ablation therapy, tumors eventually progress to a castrate-resistant stage in which the AR remains active. The mechanisms are poorly understood but it may be that changes in levels or activity of AR coregulators affect trafficking and activation of the receptor. A key stage in AR signaling occurs in the cytoplasm, where unliganded receptor is associated with the heat shock protein (HSP)90 foldosome complex. p23, a key component of this complex, is best characterized as a cochaperone for HSP90 but also has HSP90-independent activity and has been reported as having differential effects on the activity of different steroid receptors. Here we report that p23 increases activity of the AR, and this appears to involve steps both in the cytoplasm (increasing ligand-binding capacity, possibly via direct interaction with AR) and the nucleus (enhancing AR occupancy at target promoters). We show, for the first time, that AR and p23 can interact, perhaps directly, when HSP90 is not present in the same complex. The effects of p23 on AR activity are at least partly HSP90 independent because a mutant form of p23, unable to bind HSP90, nevertheless increases AR activity. In human prostate tumors, nuclear p23 was higher in malignant prostate cells compared with benign/normal cells, supporting the utility of p23 as a therapeutic target in prostate cancer.