To increase the chemical space around the well-known GalNAc-ligand as ASGPR-binder, a high-throughput screening campaign was performed, testing approximately 550,000 compounds. After evaluation of the potential screening hits, only one compound, which showed high similarity with guanosine nucleosides, was chosen for further profiling. Crystal structure analysis revealed the coordination of the Ca2+-ion within the ASGPR-binding site by the cis-diol motif of the ribose unit as well as an additional π-π-interaction of the purine heterocycle to tryptophan-243. Based on these findings, guanosine was attached via the 5'-OH group to a recently described morpholino-based nucleotide using two different linker units. The resulting morpholino-guanosine building blocks were conjugated to the 5'-end of a literature-known transthyretin targeting small interfering RNA (siRNA), leading to trivalent siRNA-guanosine conjugates, which were tested for their TTR knockdown and exhibited similar potencies as the analogous GalNAc-conjugates in vitro and in vivo.

Supplementary Data 1. Excel sheet with fold change in gene expression following anti-miR-21 treatment.

Supplementary Figure 1. miR-21 mimic suppresses expression of ANKRD46, DDAH1 and RECK. SKHep1 cells were transfected with miR-21 or negative mimic. RNA was isolated and expression of ANKRD46, DDAH1 and RECK was assessed by qPCR. (Mean, {plus minus} SD, n=3).

Supplementary Figure S1 shows that RIG-I, MDA and IRF7 mRNA expression are correlated to the Type I IFN signature in melanoma, while MAVS, TBK1 and IRF3 are not. Supplementary Figure S2 shows a list of gene products correlated with RIG-I expression based on TCGA RNAseq data from 384 melanomas. Ranking is done based on Person correlation coefficients. Supplementary Figure S3 shows a graphical illustration of RIG-I and CXCL-10 expression correlation based on TCGA RNAseq data of melanoma samples. Supplementary Figure S4 shows the results of the analysis of mRNA expression of IFN-β, RIG-I and IP-10 following RNA treatment of C8161 parental cells and CRISPR/Cas generated KO clones. Cells were treated with RIG-I agonist ppp-RNA and respective controls at 100nM. Relative mRNA levels were determined by RT-qPCR 48 hours post treatment by reverse transfection with synthetic ppp-RNA and respective OH-RNA controls as indicated. RIG-I knockout variants are characterized by absence of RIG-I agonist triggered mRNA upregulation secretion. Error bars represent standard deviation. Supplementary Figure S5 shows the flow cytometry analysis of surface antigen expression following ppp-RNA treatment of parental and KO variants of C8161 (a-c) and B16-F10 (d-f). Cells were treated with ppp-RNA and respective controls as indicated at 100nM and analyzed 48 hours post treatment. Supplementary Figure S6 shows IP 10 quantification results of C57BL/6 female mice (n=4 per group), carrying Hepa1-6 liver tumors treated with 50ug RNA complexed with In vivo Jet-PEI (i.v.). Serum was collected for IP-10 quantification 4h post RNA delivery. Error bars indicate standard deviation. Supplementary Figure S7 shows the serum cytokine response profiles 6 hours post first intratumoral treatment with RNA as indicated. Error bars reflect standard deviation. Supplementary Figure S8 shows flow cytometry based analysis of immune cell populations isolated from spleens of B16-F10 tumor bearing mice treated intratumorally with ppp-RNA or respective controls. Spleens were harvested 48h after last treatment. Error bars indicate standard deviation. Supplementary Figure S9 shows the serum cytokine response profile 6 hours post first intratumoral treatment with RNA as indicated. Error bars represent standard deviation. Supplementary Table S1 shows a list of genes comprising the type I IFN signature Supplementary Table S2 shows the results of a Pearson Correlation analysis of OVA-specific CTL frequency versus cytokine expression in the respective animals

Supplementary Figure 4. Pathway analysis of gene expression changes following anti-miR-21 treatment. SKHep1 cells treated with anti-miR-21 were subjected to microarray gene expression analysis. Changes in cellular processes (top) and pathways (bottom) upon anti-miR-21 treatment are shown.

Supplementary Figure 2. Anti-miR-21 treatment causes limited caspase 3/7 activation in non-transformed cell lines. Caspase 3/7 activation after treatment of SKHep1, IMR-90, and WI-38 cells with MM control or anti-miR-21 for 72 hours. (Mean, {plus minus} SEM, n=3).

Supplementary Tables 1 and 2. Supplementary Table 1: Fold de-repression of miR-21 target genes after anti-miR-21 treatment. Supplementary Table 2: Taqman Primer and Probes

Supplementary Figure Legends. Figure legends for Supplementary Figures 1, 2, 3, and 4.

Supplementary Figure 3. HMBG1 and LDH are induced upon miR-21 inhibition. SKHep1, HepG2 and Hep3B cells were treated with anti-miR-21 or MM control and extracellular HMBG1 and LDH activity was quantified. (Mean, {plus minus} SEM, n=3)

To expand the applicability of recently developed dioxane- and morpholino-based nucleotide analogues, their seed region destabilizing properties in small interfering RNAs (siRNAs) were investigated in order to improve potential off-target profiles. For this purpose, the corresponding adenosine analogues were synthesized in two diastereomeric series as building blocks for the automated oligonucleotide synthesis. The obtained nucleotide precursors were integrated at position 7 of an siRNA antisense strand, targeting transthyretin messenger RNA. Evaluation of the melting temperatures revealed significant differences in the obtained duplex stabilities between the two diastereomeric series, while the influence of the central scaffold was small. All siRNAs containing these novel nucleotide structures showed improved off-target profiles in vitro compared to their parent sequence with the common 2'-OMe-modified adenosine at the same position. In contrast, in vivo potencies were highly dependent on the chirality within the six-membered nucleotide scaffolds and showed high mRNA downregulations for the (2R,6R)-configured diastereomers.

Targeted extrahepatic delivery of siRNA remains a challenging task in the field of nucleic acid therapeutics. An ideal delivery tool must internalize siRNA exclusively into the cells of interest without affecting the silencing activity of siRNA. Here, we report the use of anti-EGFR Nanobodies (trademark of Ablynx N.V.) as tools for targeted siRNA delivery. A straightforward procedure for site-specific conjugation of siRNA to an engineered C-terminal cysteine residue on the Nanobody (trademark of Ablynx N.V.) is described. We show that siRNA-conjugated Nanobodies (Nb-siRNA) retain their binding to EGFR and enter EGFR-positive cells via receptor-mediated endocytosis. The activity of Nb-siRNAs was assessed by measuring the knockdown of a housekeeping gene (AHSA1) in EGFR-positive and EGFR-negative cells. We demonstrate that Nb-siRNAs are active in vitro and induce mRNA cleavage in the targeted cell line. In addition, we discuss the silencing activity of siRNA conjugated to fused Nbs with various combinations of EGFR-binding building blocks. Finally, we compare the performance of Nb-siRNA joined by four different linkers and discuss the advantages and limitations of using cleavable and noncleavable linkers in the context of Nanobody-mediated siRNA delivery.

A morpholine-based nucleotide analog was developed as a building block for hepatic siRNA targeting and stabilization. Attachment of an asialoglycoprotein-binding GalNAc ligand at the morpholine nitrogen was realized with different linkers. The obtained morpholino GalNAc scaffolds were coupled to the sense strand of a transthyretin-targeting siRNA and tested for their knockdown potency in vitro and in vivo. A clear structure-activity relationship was developed with regard to the linker type and length as well as the attachment site of the morpholino GalNAc moieties at the siRNA sense strand. Further, simple alkylation of the morpholine nitrogen led to a nucleotide analog, which increased siRNA stability, when used as a double 3'-overhang at the sense strand sequence. Combination of the best morpholino GalNAc building blocks as targeting nucleotides with an optimized stabilizing alkyl-substituted morpholine as 3'-overhangs resulted in siRNAs without any phosphorothioate stabilization in the sense strand and clearly improved the duration of action in vivo.

A novel class of nucleotide analogues with a dioxane ring as central scaffold has been developed. Synthetic routes in two diastereomeric series were realized, and the final thymidine analogues were synthesized with common functionalities for the automated oligonucleotide synthesis. The chemical space of the initially derived nucleotides was expanded by changing the central dioxane to analogous morpholine derivatives. This opens up the possibility for further derivatization by attaching different substituents at the morpholine nitrogen. The novel nucleotide building blocks were incorporated into double-stranded RNA sequences, and their hybridization properties investigated by melting-temperature analysis. Both scaffolds, dioxanes and morpholines, had an equal impact on double-strand stability, but T-m values differed depending on the chirality in the six-membered ring.

RIG-I is a highly important cytosolic pattern recognition receptor (PRR) involved in sensing RNA virus infection and inducing interferon (IFN) production. RIG-I’s natural ligand, triphosphate RNA (ppp-RNA), is proposed to be a valuable addition to the growing arsenal of cancer immunotherapy treatment options. This study validates the use of intratumoral treatment with synthetic RIG-I agonist ppp-RNA for the therapy of human cancer, with melanoma as potential entry indication amenable to intratumoral treatment. Firstly, we demonstrate that RIG-I expression is closely correlated to cellular and cytokine immune activation in a wide variety of tumor types. Secondly, cellular models of human melanoma confirm susceptibility of cancer cells to ppp-RNA treatment, revealing unexpected heterogeneity between cell lines in their selectivity for RNA features, including sequence, secondary structures and presence of triphosphate. Cellular RNA treatment responses (type I IFN, FasR, MHC-I, cytotoxicity) were demonstrated to be RIG-I dependent using RIG-I KO cells. Thirdly, we show that ppp-RNA treatment of a mouse melanoma tumor model, leads to significant local and systemic anti-tumor effects and survival benefits, associated with a type I IFN response, tumor cell apoptosis and innate and adaptive immune cell activation. For the first time, we demonstrate systemic presence of tumor antigen specific CTLs following treatment with RIG-I agonist. Overall our study demonstrates that ppp-RNA or analogs thereof have the potential to play an important role for cancer treatment in the next wave of immunotherapy. However, potential challenges in the generation and formulation of potent RIG-I agonists remain to be solved. Citation Format: Mike W. Helms, Eric Parmantier, Kerstin Jahn-Hofmann, Felix Gnerlich, Lars König, Christiane Metz-Weidmann, Monika Braun, Gabriele Dietert, Kaj Grandien, Joachim Theilhaber, Hui Cao, Tim Wagenaar, Max Schnurr, Stefan Endres, Dmitri Wiederschain, Sabine Scheidler, Simon Rothenfusser, Bodo Brunner. Treatment with synthetic RIG-I agonist triphosphate RNA leads to local and systemic anti-tumor effects in a mouse melanoma tumor model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 5024.

AbstractThe pattern recognition receptor RIG-I plays an important role in the recognition of nonself RNA and antiviral immunity. RIG-I's natural ligand, triphosphate RNA (ppp-RNA), is proposed to be a valuable addition to the growing arsenal of cancer immunotherapy treatment options. In this study, we present comprehensive data validating the concept and utility of treatment with synthetic RIG-I agonist ppp-RNA for the therapy of human cancer, with melanoma as potential entry indication amenable to intratumoral treatment. Using mRNA expression data of human tumors, we demonstrate that RIG-I expression is closely correlated to cellular and cytokine immune activation in a wide variety of tumor types. Furthermore, we confirm susceptibility of cancer cells to ppp-RNA treatment in different cellular models of human melanoma, revealing unexpected heterogeneity between cell lines in their susceptibility to RNA agonist features, including sequence, secondary structures, and presence of triphosphate. Cellular responses to RNA treatment (induction of type I IFN, FasR, MHC-I, and cytotoxicity) were demonstrated to be RIG-I dependent using KO cells. Following ppp-RNA treatment of a mouse melanoma model, we observed significant local and systemic antitumor effects and survival benefits. These were associated with type I IFN response, tumor cell apoptosis, and innate and adaptive immune cell activation. For the first time, we demonstrate systemic presence of tumor antigen–specific CTLs following treatment with RIG-I agonists. Despite potential challenges in the generation and formulation of potent RIG-I agonists, ppp-RNA or analogues thereof have the potential to play an important role for cancer treatment in the next wave of immunotherapy.

Introduction: Hepatocellular carcinoma (HCC) is the 3rd leading cause of cancer-related mortality. Currently there are few treatment options available for advanced HCC patients. Multi-kinase inhibitors sorafenib and regorafenib are the only approved systemic HCC therapies and are only marginally effective in extending survival. HCC is largely driven by difficult-to-drug oncogenes, including β-catenin, and none of the current HCC therapeutics on the market or in development addresses these major genetic drivers of disease. β-catenin is an integral part of the WNT signaling pathway and plays a major role in regulation of cell survival, apoptosis and developmental processes. The goal of this study was to systematically interrogate contribution of β-catenin to HCC pathogenesis. Methods: The extent of genomic alterations in β-catenin itself or components of its regulatory network was interrogated in two large HCC data sets, The Cancer Genome Atlas and HCC cohort from Asan Medical Center. β-catenin in vitro credentialing (target knock-down, PD modulation and cell growth inhibition) was carried out in cultured HCC cell lines using multiple siRNAs against β-catenin. Stably expressed doxycycline-inducible shRNAs targeting β-catenin were used to establish in vivo dependency in HCC tumor xenografts. Results: Building on previous observations, we detected high frequency of somatic mutations in CTNNB1/β-catenin itself and in constituent members of its regulatory network in two independent cohorts of HCC patients. Furthermore, significant upregulation in β-catenin protein levels was detected in the overwhelming majority of HCC patient samples, patient-derived xenografts and established cell lines. Using genetic tools validated for target specificity through phenotypic rescue experiments, we discovered that dependency on β-catenin in human HCC cell lines generally tracks with its activation status. HCC cell lines that harbored activating mutations in CTNNB1/β-catenin or displayed elevated levels of non-phosphorylated (active) β-catenin were significantly more sensitive to β-catenin siRNA treatment than cell lines with wild-type CTNNB1/β-catenin and lower active β-catenin. Finally, significant therapeutic benefit of β-catenin knock-down was demonstrated in established HCC tumor xenografts using doxycycline-inducible shRNA system. β-catenin downregulation and tumor growth inhibition was associated with reduction in PD marker AXIN2, and decreased cancer cell proliferation. Conclusions: β-catenin is an important therapeutic target in HCC that could potentially be targeted using therapeutic siRNA suitably formulated for delivery to human liver tumors. Genomic alterations in CTNNB1/β-catenin, as well as increased abundance of non-phosphorylated (active) β-catenin, may serve as predictive biomarkers for patient selection. Citation Format: Zhihu (Jeff) Ding, Chaomei Shi, Lan Jiang, Tatiana Tolstykh, Hui Cao, Dinesh Bangari, Susan Ryan, Taiguang Jin, Mikhail Levit, Karl Mamaat, Qunyan Yu, Hui Qu, Joern Hopke, May Cindhuchao, Dietmar Hoffmann, Fangxian Sun, Mike Helms, Kerstin Jahn-Hofmann, Sabine Scheidler, Douglas Fang, Liang Schweizer, Jack Pollard, Christopher Winter, Dmitri Wiederschain. Sensitivity of liver cancer cell lines to B-catenin knock-down correlates with pathway activation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2489.

Hepatocellular carcinoma (HCC) represents a serious public health challenge with few therapeutic options available to cancer patients.Wnt/β-catenin pathway is thought to play a significant role in HCC pathogenesis. In this study, we confirmed high frequency of CTNNB1 (β-catenin) mutations in two independent cohorts of HCC patients and demonstrated significant upregulation of β-catenin protein in the overwhelming majority of HCC patient samples, patient-derived xenografts (PDX) and established cell lines. Using genetic tools validated for target specificity through phenotypic rescue experiments, we went on to investigate oncogenic dependency on β-catenin in an extensive collection of human HCC cells lines. Our results demonstrate that dependency on β-catenin generally tracks with its activation status. HCC cell lines that harbored activating mutations in CTNNB1 or displayed elevated levels of non-phosphorylated (active) β-catenin were significantly more sensitive to β-catenin siRNA treatment than cell lines with wild-type CTNNB1 and lower active β-catenin. Finally, significant therapeutic benefit of β-catenin knock-down was demonstrated in established HCC tumor xenografts using doxycycline-inducible shRNA system. β-catenin downregulation and tumor growth inhibition was associated with reduction in AXIN2, direct transcriptional target of β-catenin, and decreased cancer cell proliferation as measured by Ki67 staining. Taken together, our data highlight fundamental importance of aberrant β-catenin signaling in the maintenance of oncogenic phenotype in HCC.

Abstract Hepatocellular carcinoma (HCC) remains a significant clinical challenge with few therapeutic options available to cancer patients. MicroRNA 21-5p (miR-21) has been shown to be upregulated in HCC, but the contribution of this oncomiR to the maintenance of tumorigenic phenotype in liver cancer remains poorly understood. We have developed potent and specific single-stranded oligonucleotide inhibitors of miR-21 (anti-miRNAs) and used them to interrogate dependency on miR-21 in a panel of liver cancer cell lines. Treatment with anti–miR-21, but not with a mismatch control anti-miRNA, resulted in significant derepression of direct targets of miR-21 and led to loss of viability in the majority of HCC cell lines tested. Robust induction of caspase activity, apoptosis, and necrosis was noted in anti–miR-21-treated HCC cells. Furthermore, ablation of miR-21 activity resulted in inhibition of HCC cell migration and suppression of clonogenic growth. To better understand the consequences of miR-21 suppression, global gene expression profiling was performed on anti–miR-21-treated liver cancer cells, which revealed striking enrichment in miR-21 target genes and deregulation of multiple growth-promoting pathways. Finally, in vivo dependency on miR-21 was observed in two separate HCC tumor xenograft models. In summary, these data establish a clear role for miR-21 in the maintenance of tumorigenic phenotype in HCC in vitro and in vivo. Implications: miR-21 is important for the maintenance of the tumorigenic phenotype of HCC and represents a target for pharmacologic intervention. Mol Cancer Res; 13(6); 1009–21. ©2015 AACR.

Hepatocellular carcinoma (HCC) remains a significant unmet medical need with few therapeutic options available. Micro RNA 21 (miR-21) has been shown to be upregulated in HCC, however, contribution of this onco-miR to the maintenance of tumorigenic phenotype in liver cancer remains poorly understood. We have developed potent and specific single-stranded oligonucleotide inhibitors of miR-21 (anti-miR-21) and used them to interrogate dependency on miR-21 in a panel of 20 commercially available HCC cell lines in vitro. Upon lipid-mediated transfection, anti-miR-21, but not its mismatched (MM) control, caused significant de-repression of known direct targets of miR-21 (ANKRD46, DDAH1, RECK1) and inhibited survival of a large subset of HCC cell lines. Treatment of these sensitive HCC cell lines with anti-miR-21 resulted in dose- and time-dependent induction of caspase 3/7 activity. In contrast, non-responder HCC cell lines failed to significantly upregulate caspase activity and maintained viability in the presence of anti-miR compound. Further analysis of responder cell lines revealed robust induction of cell death, inhibition of cell migration and suppression of clonogenic growth upon treatment with miR-21 inhibitor. To better understand the consequences of miR-21 suppression in HCC, we carried out global gene expression profiling of anti-miR-21 treated sensitive liver cancer cells. Striking enrichment in miR-21 targets was noted among upregulated transcripts. Gene ontology analysis identified key cellular processes affected by miR-21 inhibition, including deregulation of metabolic pathways. In addition to the induction of direct miR-21 targets, cyclin H was found to be significantly downregulated upon miR-21 inhibition in the majority of responder cell lines. We hypothesize that inhibition of cyclin H expression, while an indirect effect of miR-21 suppression, could contribute to the activity of anti-miR-21 compounds. In summary, our data suggest that inhibition of miR-21 merits further investigation in the treatment of hepatocellular carcinoma. Citation Format: Sonya Zabludoff, Timothy Wagenaar, Francisco Adrian, Charles Allerson, Heike Arlt, Raffaele Baffa, Bal Bhat, Hui Cao, Scott Davis, Carlos Garcia-Echeverria, Kathrin Heermeier, Shih-Min Huang, Lan Jiang, Eric Marcusson, Christiane Metz-Weidmann, Adam Pavlicek, Jack Pollard, Jennifer Rocnik, Sabine Scheidler, Chaomei Shi, Fangxian Sun, Tatiana Tolstykh, Qunyan Yu, Gang Zheng, Dmitri Wiederschain. Targeting miR-21 in hepatocellular carcinoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4787. doi:10.1158/1538-7445.AM2014-4787

Abstract: In renal HEK‐293 cells, the dietary Maillard reaction compounds casein‐linked Nε‐carboxymethyllysine (CML), CML, bread crust (BC), and pronyl‐glycine (a key compound formed in association with the process‐induced heat impact applied to bread dough) all showed activation of p38‐MAP kinase. Expression of the C‐terminus truncated receptor for advanced glycation end products (RAGE) resulted in a reduction of HEK‐293‐MAP kinase activation. As these findings suggested a RAGE‐mediated activating effect of CML, BC, and pronyl‐glycine on kidney cellular signal transduction pathways, an in vivo study was performed. Male Wistar rats were subjected to a sham operation (CTRL, n= 20) or to 5/6 nephrectomy (NX, n= 20). Both groups were randomized into two subgroups and fed 20 g of a diet containing either 25% by weight BC or wheat starch (WS). GC‐MS analyses of CML, carboxyethyllysine (CEL), and pentosidine revealed increased levels of CML and CEL in the liver but decreased levels of CML in the kidneys of CTRL and NX rats fed the BC diet compared to those on the WS diet. However, urinary levels of CML were also elevated in the CTRL and NX rats on the BC diet, pointing to enhanced excretion of AGEs after BC administration. Although renal insufficiency in the NX rats was reflected by proteinuria, the renal handling of CML and, presumably, other AGEs was not impaired.