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Supplementary Figure 1. Characterization of inflammatory cell infiltrates and their recovery with time. Supplementary Figure 2. Negative feedback control of pTBK1 by autophagy. Supplementary Figure 3. pTBK1 levels correlate with underlying levels of basal autophagy. Supplementary Figure 4. Lack of total TBK1 enrichment in autophagosomes. Supplementary Figure 5. Screening of autophagy receptors that regulate selective autophagy of pTBK1. Supplementary Figure 6. MRT67307 or CYT387 treatment inhibits pathogen xenophagy. Supplementary Figure 7. CYT387 treatment inhibits IFNγ induced PD-L1 expression in 8988T cells. Supplementary Figure 8. Jurkat T cell recruitment by exogenous CCL5 or 8988TsgATG5 cells.

Autophagy promotes tumor progression downstream of oncogenic KRAS, yet also restrains inflammation and dysplasia through mechanisms that remain incompletely characterized. Understanding the basis of this paradox has important implications for the optimal targeting of autophagy in cancer. Using a mouse model of cerulein-induced pancreatitis, we found that loss of autophagy by deletion of Atg5 enhanced activation of the IκB kinase (IKK)-related kinase TBK1 in vivo, associated with increased neutrophil and T-cell infiltration and PD-L1 upregulation. Consistent with this observation, pharmacologic or genetic inhibition of autophagy in pancreatic ductal adenocarcinoma cells, including suppression of the autophagy receptors NDP52 or p62, prolonged TBK1 activation and increased expression of CCL5, IL6, and several other T-cell and neutrophil chemotactic cytokines in vitro. Defective autophagy also promoted PD-L1 upregulation, which is particularly pronounced downstream of IFNγ signaling and involves JAK pathway activation. Treatment with the TBK1/IKKϵ/JAK inhibitor CYT387 (also known as momelotinib) not only inhibits autophagy, but also suppresses this feedback inflammation and reduces PD-L1 expression, limiting KRAS-driven pancreatic dysplasia. These findings could contribute to the dual role of autophagy in oncogenesis and have important consequences for its therapeutic targeting. Cancer Immunol Res; 4(6); 520–30. ©2016 AACR.

In order to achieve efficient therapeutic post-transcriptional gene-silencing mediated by the RNA interference (RNAi) pathway, small interfering RNAs (siRNAs) must be chemically modified. Several supra-RNA structures, with the potential to stabilize siRNAs metabolically have been evaluated for their ability to induce gene silencing, but all have limitations or have not been explored in therapeutically relevant contexts. Covalently closed circular RNA transcripts are prevalent in eukaryotes and have potential as biomarkers and disease targets, and circular RNA mimics are being explored for use as therapies. Here we report the synthesis and evaluation of small circular interfering RNAs (sciRNAs). To synthesize sciRNAs, a sense strand functionalized with the trivalent N-acetylgalactosamine (GalNAc) ligand and cyclized using ‘click’ chemistry was annealed to an antisense strand. This strategy was used for synthesis of small circles, but could also be used for synthesis of larger circular RNA mimics. We evaluated various sciRNA designs in vitro and in vivo. We observed improved metabolic stability of the sense strand upon circularization and off-target effects were eliminated. The 5′-(E)-vinylphosphonate modification of the antisense strand resulted in GalNAc-sciRNAs that are potent in vivo at therapeutically relevant doses. Physicochemical studies and NMR-based structural analysis, together with molecular modeling studies, shed light on the interactions of this novel class of siRNAs, which have a partial duplex character, with the RNAi machinery.

Preclinical mechanistic studies have pointed towards RNA interference-mediated off-target effects as a major driver of hepatotoxicity for GalNAc–siRNA conjugates. Here, we demonstrate that a single glycol nucleic acid or 2′–5′-RNA modification can substantially reduce small interfering RNA (siRNA) seed-mediated binding to off-target transcripts while maintaining on-target activity. In siRNAs with established hepatotoxicity driven by off-target effects, these novel designs with seed-pairing destabilization, termed enhanced stabilization chemistry plus (ESC+), demonstrated a substantially improved therapeutic window in rats. In contrast, siRNAs thermally destabilized to a similar extent by the incorporation of multiple DNA nucleotides in the seed region showed little to no improvement in rat safety suggesting that factors in addition to global thermodynamics play a role in off-target mitigation. We utilized the ESC+ strategy to improve the safety of ALN-HBV, which exhibited dose-dependent, transient and asymptomatic alanine aminotransferase elevations in healthy volunteers. The redesigned ALN-HBV02 (VIR-2218) showed improved specificity with comparable on-target activity and the program was reintroduced into clinical development.

In this report, we investigated the hexopyranose chemical modification Altriol Nucleic Acid (ANA) within small interfering RNA (siRNA) duplexes that were otherwise fully modified with the 2'-deoxy-2'-fluoro and 2'-O-methyl pentofuranose chemical modifications. The siRNAs were designed to silence the transthyretin (Ttr) gene and were conjugated to a trivalent N-acetylgalactosamine (GalNAc) ligand for targeted delivery to hepatocytes. Sense and antisense strands of the parent duplex were synthesized with single ANA residues at each position on the strand, and the resulting siRNAs were evaluated for their ability to inhibit Ttr mRNA expression in vitro. Although ANA residues were detrimental at the 5' end of the antisense strand, the siRNAs with ANA at position 6 or 7 in the seed region had activity comparable to the parent. The siRNA with ANA at position 7 in the seed region was active in a mouse model. An Oligonucleotide with ANA at the 5' end was more stable in the presence of 5'-exonuclease than an oligonucleotide of the same sequence and chemical composition without the ANA modification. Modeling studies provide insight into the origins of regiospecific changes in potency of siRNAs and the increased protection against 5'-exonuclease degradation afforded by the ANA modification. ispartof: NUCLEIC ACIDS RESEARCH vol:48 issue:8 pages:4028-4040 ispartof: location:England status: published

RNA interference (RNAi) therapeutics are a new class of medicines that can address unmet medical needs by silencing disease-causing gene transcripts. While delivery of short interfering RNAs (siRNAs) to hepatocytes has yielded multiple drug approvals, novel delivery solutions are needed to expand the reach of RNAi therapeutics. Here we report that conjugation of 2'-O-hexadecyl (C16) to siRNAs enables efficient silencing in the central nervous system (CNS), eye, and lung of multiple nonclinical species with broad cell type specificity. Intrathecally delivered C16-siRNAs are active across CNS regions and cell types, with sustained silencing for at least three months, which is an especially important outcome considering the challenging dosing route. Similarly, intravitreal and intranasal administration of C16-siRNAs resulted in potent and sustained knockdown in the eye and lung, respectively. Efficient delivery facilitated through C16 conjugation to optimized siRNA designs has enabled candidate selection for investigational human clinical trials assessing therapeutic silencing beyond the liver with infrequent (e.g. bi-annual) dosing.

Therapeutics based on short interfering RNAs (siRNAs) delivered to hepatocytes have been approved, but new delivery solutions are needed to target additional organs. Here we show that conjugation of 2'-O-hexadecyl (C16) to siRNAs enables safe, potent and durable silencing in the central nervous system (CNS), eye and lung in rodents and non-human primates with broad cell type specificity. We show that intrathecally or intracerebroventricularly delivered C16-siRNAs were active across CNS regions and cell types, with sustained RNA interference (RNAi) activity for at least 3 months. Similarly, intravitreal administration to the eye or intranasal administration to the lung resulted in a potent and durable knockdown. The preclinical efficacy of an siRNA targeting the amyloid precursor protein was evaluated through intracerebroventricular dosing in a mouse model of Alzheimer's disease, resulting in amelioration of physiological and behavioral deficits. Altogether, C16 conjugation of siRNAs has the potential for safe therapeutic silencing of target genes outside the liver with infrequent dosing.

We recently reported that RNAi-mediated off-target effects are important drivers of the hepatotoxicity observed for a subset of GalNAc–siRNA conjugates in rodents, and that these findings could be mitigated by seed-pairing destabilization using a single GNA nucleotide placed within the seed region of the guide strand. Here, we report further investigation of the unique and poorly understood GNA/RNA cross-pairing behavior to better inform GNA-containing siRNA design. A reexamination of published GNA homoduplex crystal structures, along with a novel structure containing a single (S)-GNA-A residue in duplex RNA, indicated that GNA nucleotides universally adopt a rotated nucleobase orientation within all duplex contexts. Such an orientation strongly affects GNA-C and GNA-G but not GNA-A or GNA-T pairing in GNA/RNA heteroduplexes. Transposition of the hydrogen-bond donor/acceptor pairs using the novel (S)-GNA-isocytidine and -isoguanosine nucleotides could rescue productive base-pairing with the complementary G or C ribonucleotides, respectively. GalNAc-siRNAs containing these GNA isonucleotides showed an improved in vitro activity, a similar improvement in off-target profile, and maintained in vivo activity and guide strand liver levels more consistent with the parent siRNAs than those modified with isomeric GNA-C or -G, thereby expanding our toolbox for the design of siRNAs with minimized off-target activity.

Hereditary angioedema (HAE) is a genetic disorder mostly caused by mutations in the C1 esterase inhibitor gene (C1INH) that results in poor control of contact pathway activation and excess bradykinin generation. Bradykinin increases vascular permeability and is ultimately responsible for the episodes of swelling characteristic of HAE. We hypothesized that the use of RNA interference (RNAi) to reduce plasma Factor XII (FXII), which initiates the contact pathway signaling cascade, would reduce contact pathway activation and prevent excessive bradykinin generation. A subcutaneously administered GalNAc-conjugated small-interfering RNA (siRNA) targeting F12 mRNA (ALN-F12) was developed, and potency was evaluated in mice, rats, and cynomolgus monkeys. The effect of FXII reduction by ALN-F12 administration was evaluated in two different vascular leakage mouse models. An ex vivo assay was developed to evaluate the correlation between human plasma FXII levels and high-molecular weight kininogen (HK) cleavage. A single subcutaneous dose of ALN-F12 led to potent, dose-dependent reduction of plasma FXII in mice, rats, and NHP. In cynomolgus monkeys, a single subcutaneous dose of ALN-F12 at 3 mg/kg resulted in >85% reduction of plasma FXII. Administration of ALN-F12 resulted in dose-dependent reduction of vascular permeability in two different mouse models of bradykinin-driven vascular leakage, demonstrating that RNAi-mediated reduction of FXII can potentially mitigate excess bradykinin stimulation. Lastly, ex vivo human plasma HK cleavage assay indicated FXII-dependent bradykinin generation. Together, these data suggest that RNAi-mediated knockdown of FXII by ALN-F12 is a potentially promising approach for the prophylactic treatment of HAE.

Small interfering RNAs (siRNAs) conjugated to a trivalent N-acetylgalactosamine (GalNAc) ligand are being evaluated in investigational clinical studies for a variety of indications. The typical development candidate selection process includes evaluation of the most active compounds for toxicity in rats at pharmacologically exaggerated doses. The subset of GalNAc-siRNAs that show rat hepatotoxicity is not advanced to clinical development. Potential mechanisms of hepatotoxicity can be associated with the intracellular accumulation of oligonucleotides and their metabolites, RNA interference (RNAi)-mediated hybridization-based off-target effects, and/or perturbation of endogenous RNAi pathways. Here we show that rodent hepatotoxicity observed at supratherapeutic exposures can be largely attributed to RNAi-mediated off-target effects, but not chemical modifications or the perturbation of RNAi pathways. Furthermore, these off-target effects can be mitigated by modulating seed-pairing using a thermally destabilizing chemical modification, which significantly improves the safety profile of a GalNAc-siRNA in rat and may minimize the occurrence of hepatotoxic siRNAs across species., A subset of chemically-modified siRNAs conjugated to trivalent GalNAc may fail during nonclinical development due to rat hepatotoxicity. Here, the authors show that hepatotoxicity may be accounted for by microRNA-like off-target effects of siRNA and can be mitigated by a thermally destabilizing modification in the siRNA seed region.

We report rapid, potent reversal of GalNAc-siRNA-mediated RNA interference (RNAi) activity in vivo with short, synthetic, high-affinity oligonucleotides complementary to the siRNA guide strand. We found that 9-mers with five locked nucleic acids (LNAs) have the highest potency across several targets. Our modular, sequence-specific approach, named REVERSIR, may enhance the therapeutic profile of any long-acting GalNAc-siRNA (short interfering RNA) conjugate by enabling control of RNAi pharmacology.

Using differential display, we identified the Anabaena sp. PCC 7120 ribulose 1,5-bisphosphate carboxylase transcriptional regulator (rbcR1) gene, a member of the LysR family of positive transcription factors. The rbcR1 transcript and its putative target gene ribulose 1,5-bisphosphate carboxylase/oxygenase (rbcL/S) were repressed by cold (20 degrees C) and osmotic (sucrose and salt) stress. Cold stress also induced a transient downregulation of the Anabaena 7120 ntcA transcriptional regulator. Expression of the ntcA gene, however, returned to normal levels 2 h after initiation of cold stress and increased significantly above normal levels 24 h after growth at 20 degrees C. The early decline in the expression of the ntcA, rbcR1, and rbcL/S transcripts appears to be part of the Anabaena 7120 global adaptation response to stress. The substantial increase in the ntcA gene expression 24 h following cold stress suggests that Anabaena 7120 experiences substantial nitrogen limitation under these conditions. These data suggest that in response to stress, Anabaena 7120 decreases its metabolic activity through regulation of the CO(2) fixation machinery while enhancing its nitrogen assimilation by inducing the expression of the nitrogen global transcriptional regulator, NtcA.