Small activating RNAs (saRNAs) are short, double-stranded oligonucleotides designed to specifically upregulate target mRNA and protein expression by loading into an Ago2 complex for nuclear translocation and activation of RNA polymerase II activity. We have developed MTL-CEBPA, an saRNA-based therapeutic designed to increase the expression of the CEBPA transcription factor that acts as both a tumor suppressor and a key modulator of the tumor microenvironment. In preclinical tumor models MTL-CEBPA has single-agent antitumor activity and very significantly improves the activity of both sorafenib and checkpoint inhibitors through impacting the immunosuppressive tumor microenvironment. This is the first saRNA drug to enter clinical trials, and we have been investigating whether this technology could be extended to activate endogenous immune stimulatory genes for utilization in cancer immunotherapy. We designed saRNA to target the IL-23A, IL-36g, OX40L, and UCP2 transcripts using our proprietary algorithm that bind to long noncoding RNA at position +2000 to -2000 nucleotides relative to the transcriptional start site. At least five 19-nucleotide sequences from the algorithm for each target were synthesized with mUmU overhangs at the 3´ ends of both strands to improve stability and reduce nonspecific immunogenicity. The saRNAs were screened by transfection in human (HepG2 and A549) or murine (CT26, BNL.1ME and RAW264.7) cells, and mRNA levels of the target genes were measured by qPCR. The screening process identified active saRNAs for all 4 targets leading to upregulation of target mRNA by 1.9 to 6.7-fold at 48-72 hours post transfection compared to oligonucleotide transfection controls. Sequence-dependent specificity was confirmed by modifying 2 to 3 nucleotide residues in the seed binding region of the lead sequences, which in each case significantly reduced induction of target mRNA compared to the parental saRNA sequences. For each lead saRNA, increased target protein was also confirmed either by immunofluorescence or ELISA and increased functional activity was confirmed either by impact on downstream targets or phenotypic activity. For example, overexpression of UCP2 transgene in tumor cell lines has been demonstrated by others to suppress tumor cell proliferation and imitate an antitumor immune cycle. We showed that these effects were recapitulated in vitro by increasing endogenous UCP2 expression using saRNA. Here, we demonstrate the utilization of saRNA technology to upregulate immune stimulatory targets for immunotherapy. We are currently progressing efficacy studies in a number of preclinical tumor models.

Citation Format: Choon Ping Tan, Laura Sinigaglia, Siv A Hegre, Alexandre Debacker, Albert Kwok, Pal Saetrom, Matthew Catley, David C. Blakey, Nagy Habib. Novel approach for upregulation of endogenous immunostimulatory targets for cancer therapy [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2019 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(3 Suppl):Abstract nr A72.