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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-1β, 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., MTL-CEBPA is a small activating RNA that targets the upregulation of transcription factor C/EBPα, currently being evaluated in a phase 1b trial for treatment of hepatocellular carcinoma. Zhou et al. demonstrate that MTL-CEBPA increases the expression of C/EBPα and displays anti-inflammatory effects in a tissue culture system and a humanized mouse model.

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.

Aptamer Drug Conjugates (ApDCs) are a potential class of targeting therapeutics to improve therapeutic index over traditional chemotherapy. As most existing chemotherapies are absorbed into cells non-specifically through lipophilic interaction, to improve the therapeutic index, the drugs were attached to pancreatic cancer specific RNA aptamer, P19, as targeting moieties that preferentially delivered the payload to tumor cells. Active metabolites of gemcitabine (dFdCMP) and 5FdU (5FdUMP) were incorporated P19 intrinsically to make conjugates. The conjugates of P19-dFdCMP or P19-5FdUMP kept their structural functionality getting internalized in their target cells, PANC-1. P19-dFdCMP and P19-5FdUMP was incorporated into replicating DNA, resulting in chain termination and stalling of replication forks in nuclear. They induced the phosphorylation of histone H2AX on Ser139 (γ-H2AX), markers of double-strand DNA breaks, and increased the forms of nuclear foci at the sites of DNA damage. In consequence, the cell proliferation was significantly inhibited 51-53% in PANC-1 and 54-34% in gemcitabine resistance AsPC-1. Targeted anti-mitotic therapies have emerged as a new concept of cancer drugs. Chemotoxine, monomethyl auristatin E (MMAE) is a very potent antimitotic agent that inhibits cell division by blocking the polymerization of tubulin. However, it can't be used by drug itself because of high toxicity. Herein, toxic molecule, MMAE, was conjugated to P19 for the targeted anti-mitotic therapies. P19-MMAE caused mitotic G2/M phase arrest, consequently inhibited 56% of cell proliferation on dose-dependent manner comparing control. In vivo assay, P19-MMAE induced the tumor regression via tail vein injection. In this study, the drug attached aptamer significantly decreased non-specific uptake of the drug and increase specific uptake of the conjugate in tumor cells. Our approaches suggest guiding cytotoxic drugs into malignant cancer cells specifically without causing significant harm to the normal cells.

Transcriptional factor C/EBPα (CCAAT/enhancer-binding protein-a) is downregulated and silenced in pancreatic ductal adenocarcinoma (PDAC). In this study, we tested the feasibility of targeting promoter regions of transcriptional factor C/EBPα to induce transcriptional activation by small activation RNA (saRNA) for cancer therapeutics in PDAC. C/EBPα is known to up-regulate p21, a strong inhibitor of cell proliferation. C/EBPα-saRNA inhibited cell proliferation in PDAC. For the targeted delivery into cells, small structured single-stranded RNAs known as aptamers, recognizing targets specifically, were employed for the effective delivering vehicles. 2’-fluropyrimidine RNA aptamers (2’F-RNA) were selected to acquire nuclease resistance based on the whole cell SELEX. The cell internalization was evaluated through flow cytometry and confocal microscopy. The selected 2’F-RNA aptamers got internalized into a variety of pancreatic cancer cell lines. Human pancreatic cancer specimen histopathology and cell binding assays indicated that they recognized cancer cells specifically, not to normal cells. The conjugates, aptamer with C/EBPα-saRNA, were constructed for the targeted delivery. The conjugates escaped endosome and localized in nuclear. The conjugates of aptamer with C/EBPα-saRNA inhibited cell proliferation and upregulated gene expression. Xenograft mice model was used to test tumor cell growth inhibition in vivo throughout the transcriptional activation of C/EBPα. The conjugates of aptamer with C/EBPα-saRNA reduced tumor burden significantly in vivo, showing its anti-tumor effects. The aptamer target expressed on cell membrane was identified mitochondrial Hsp70. For the first time, results from these studies provide a new therapeutics strategy through transcriptional activation of C/EBPα by aptamer mediation induce anti-tumor effects in pancreatic cancer.