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

Abstract

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.

Keywords: C/EBP; CEBPA; HCC; RNA activation; hepatocellular carcinoma; liver cancer; saRNA; small RNA.

Figure 1

Nucleotide Walk on CEBPA saRNA Hotspots in HepG2 Cells (A) Schematic showing location and orientation of CEBPA mRNA and antisense transcript GenBank: AW665812, with approximate locations of AW1 and AW2 hotspots. (B) Expression of CEBPA mRNA for each sequence transfected at 50 nM relative to mock transfected cells. (C) Expression of albumin mRNA for each sequence transfected at 50 nM relative to mock transfected cells. Error bars represent SEM.

Figure 2

Mechanism of Action of AW1-51 saRNA in HepG2 Cells (A) Relative luciferase activity representing C/EBP protein activity after transfection with 10 nM AW1-51 saRNA. (B) qPCR for CEBPA mRNA on nascent transcripts isolated from nuclear run-on after 10 nM AW1-51 saRNA transfection. (C) qPCR for CEBPA mRNA after transfection with 10 nM AW1-51 duplexes with a 5′ inverted abasic modification on the indicated strand. (D) qPCR for CEBPA mRNA after transfection with 10 nM AW1-51 saRNA with mutations in the seed region. (E) qPCR for CEBPA mRNA after transfection with 10 nM AW1-51 saRNA with mutations in the center of the duplex. (F) qPCR for GenBank: AW665812 RNA after transfection with 10 nM AW1-51. Statistical significance shown for the indicated condition compared to NC transfection: *p < 0.05; **p < 0.01; ***p < 0.001. Error bars represent SEM.

Figure 3

Activity of CEBPA-51 in the HCC Lines HepG2 and Hep3B (A) qPCR for CEBPA and ALB mRNA after transfection with 10 nM CEBPA-51. (B) Western blot for C/EBP-α after transfection with 10 nM CEBPA-51. (C) WST-1 assays over a 96-hr time course after transfection with 10 nM CEBPA-51. Statistical significance shown for CEBPA-51 compared to FLUC transfection: *p < 0.05; **p < 0.01. Error bars represent SEM.

Figure 4

Mechanism of Action of CEBPA-51 in HepG2 Cells (A) Western blot after co-immunoprecipitation of Ago1–4 and biotinylated CEBPA-51. (B) qPCR for CEBPA mRNA after transfection of 10 nM CEBPA-51 wild-type and Ago2 knockout MEFs. (C) qPCR at the CEBPA locus after chromatin immunoprecipitation with biotinylated CEBPA-51. The CEBPA-51 target site is approximately 3 kb downstream of the CEBPA TSS. (D) qPCR for CTR9 mRNA after transfection with CTR9 siRNA and qPCR for CEBPA mRNA after co-transfection of CTR9 siRNA and CEBPA-51. Statistical significance shown for the indicated condition compared to FLUC transfection: **p < 0.01. Error bars represent SEM.