Polyphosphate-induced thrombosis in mice is factor XII dependent and is attenuated by histidine-rich glycoprotein.

Histidine-rich glycoprotein (HRG) is an abundant plasma protein that binds factor XIIa (FXIIa) and inhibits factor XII (FXII) autoactivation and FXIIa-mediated activation of FXI. Polyphosphate (polyP), a potent procoagulant released from activated platelets, may serve as a physiological activator of the contact system. Previously, we showed that HRG binds DNA and neutralizes its procoagulant activity. Consequently, our goal was to determine whether the capacity of HRG to bind polyanions enables it to regulate polyP-induced thrombosis. In a plate-based assay, immobilized polyP bound HRG, FXII, and FXIIa in a zinc-dependent manner. Basal and polyP-induced thrombin generation was greater in plasma from HRG-deficient mice than in plasma from wild-type mice. Intraperitoneal injection of polyP shortened the activated partial thromboplastin time, enhanced thrombin generation, increased thrombin-antithrombin levels, reduced lung perfusion, and promoted pulmonary fibrin deposition to a greater extent in HRG-deficient mice than in wild-type mice, effects that were abrogated with FXII knockdown. HRG thus attenuates the procoagulant and prothrombotic effects of polyP in an FXII-dependent manner by modulating the contact system.

Antisense Oligonucleotides Targeting Jagged 1 Reduce House Dust Mite-induced Goblet Cell Metaplasia in the Adult Murine Lung.

Goblet cell metaplasia, excessive mucus production, and inadequate mucus clearance accompany and exacerbate multiple chronic respiratory disorders, such as asthma and chronic obstructive pulmonary disease. Notch signaling plays a central role in controlling the fate of multiple cell types in the lung, including goblet cells. In the present study, we explored the therapeutic potential of modulating the Notch pathway in the adult murine lung using chemically modified antisense oligonucleotides (ASOs). To this end, we designed and characterized ASOs targeting the Notch receptors Notch1, Notch2, and Notch3 and the Notch ligands Jag1 (Jagged 1) and Jag2 (Jagged 2). Pulmonary delivery of ASOs in healthy mice or mice exposed to house dust mite, a commonly used mouse model of asthma, resulted in a significant reduction of the respective mRNAs in the lung. Furthermore, ASO-mediated knockdown of Jag1 or Notch2 in the lungs of healthy adult mice led to the downregulation of the club cell marker Scgb1a1 and the concomitant upregulation of the ciliated cell marker FoxJ1 (forkhead box J1). Similarly, ASO-mediated knockdown of Jag1 or Notch2 in the house dust mite disease model led to reduced goblet cell metaplasia and decreased mucus production. Because goblet cell metaplasia and excessive mucus secretion are a common basis for many lung pathologies, we propose that ASO-mediated inhibition of JAG1 could provide a novel therapeutic path for the treatment of multiple chronic respiratory diseases.

Steric Inhibition of 5' UTR Regulatory Elements Results in Upregulation of Human CFTR.

Cystic fibrosis (CF) is an autosomal recessive monogenic disease caused by mutations in the CFTR gene. Therapeutic approaches that are focused on correcting CFTR protein face the challenge of the heterogeneity in CFTR mutations and resulting defects. Thus, while several small molecules directed at CFTR show benefit in the clinic for subsets of CF patients, these drugs cannot treat all CF patients. Additionally, the clinical benefit from treatment with these modulators could be enhanced with novel therapies. To address this unmet need, we utilized an approach to increase CFTR protein levels through antisense oligonucleotide (ASO)-mediated steric inhibition of 5' UTR regulatory elements. We identified ASOs to upregulate CFTR protein expression and confirmed the regulatory role of the sites amenable to ASO-mediated upregulation. Two ASOs were investigated further, and both increased CFTR protein expression and function in cell lines and primary human bronchial epithelial cells with distinct CF genotypes. ASO treatment further increased CFTR function in almost all CF genotypes tested on top of treatment with the FDA approved drug Symdeko (ivacaftor and tezacaftor). Thus, we present a novel approach to CFTR therapeutic intervention, through ASO-mediated modulation of translation.

Inhibition of the extrinsic or intrinsic coagulation pathway during pneumonia-derived sepsis.

Pneumonia is the most frequent cause of sepsis, and Klebsiella pneumoniae is a common pathogen in pneumonia and sepsis. Infection is associated with activation of the coagulation system. Coagulation can be activated by the extrinsic and intrinsic routes, mediated by factor VII (FVII) and factor XII (FXII), respectively. To determine the role of FVII and FXII in the host response during pneumonia-derived sepsis, mice were treated with specific antisense oligonucleotide (ASO) directed at FVII or FXII for 3 wk before infection with K. pneumoniae via the airways. FVII ASO treatment strongly inhibited hepatic FVII mRNA expression, reduced plasma FVII to ~25% of control, and selectively prolonged the prothrombin time. FXII ASO treatment strongly suppressed hepatic FXII mRNA expression, reduced plasma FXII to ~20% of control, and selectively prolonged the activated partial thromboplastin time. Lungs also expressed FVII mRNA, which was not altered by FVII ASO administration. Very low FXII mRNA levels were detected in lungs, which were not modified by FXII ASO treatment. FVII ASO attenuated systemic activation of coagulation but did not influence fibrin deposition in lung tissue. FVII ASO enhanced bacterial loads in lungs and mitigated sepsis-induced distant organ injury. FXII inhibition did not affect any of the host response parameters measured. These results suggest that partial inhibition of FVII, but not of FXII, modifies the host response to gram-negative pneumonia-derived sepsis.

IL-4 Receptor Alpha Signaling through Macrophages Differentially Regulates Liver Fibrosis Progression and Reversal.

Chronic hepatitis leads to liver fibrosis and cirrhosis. Cirrhosis is a major cause of worldwide morbidity and mortality. Macrophages play a key role in fibrosis progression and reversal. However, the signals that determine fibrogenic vs fibrolytic macrophage function remain ill defined. We studied the role of interleukin-4 receptor α (IL-4Rα), a potential central switch of macrophage polarization, in liver fibrosis progression and reversal. We demonstrate that inflammatory monocyte infiltration and liver fibrogenesis were suppressed in general IL-4Rα-/- as well as in macrophage-specific IL-4Rα-/- (IL-4RαΔLysM) mice. However, with deletion of IL-4RαΔLysM spontaneous fibrosis reversal was retarded. Results were replicated by pharmacological intervention using IL-4Rα-specific antisense oligonucleotides. Retarded resolution was linked to the loss of M2-type resident macrophages, which secreted MMP-12 through IL-4 and IL-13-mediated phospho-STAT6 activation. We conclude that IL-4Rα signaling regulates macrophage functional polarization in a context-dependent manner. Pharmacological targeting of macrophage polarization therefore requires disease stage-specific treatment strategies. RESEARCH IN CONTEXT: Alternative (M2-type) macrophage activation through IL-4Rα promotes liver inflammation and fibrosis progression but speeds up fibrosis reversal. This demonstrates context dependent, opposing roles of M2-type macrophages. During reversal IL-4Rα induces fibrolytic MMPs, especially MMP-12, through STAT6. Liver-specific antisense oligonucleotides efficiently block IL-4Rα expression and attenuate fibrosis progression.

Inhaled ENaC antisense oligonucleotide ameliorates cystic fibrosis-like lung disease in mice.

BACKGROUND: Epithelial sodium channel (ENaC, Scnn1) hyperactivity in the lung leads to airway surface dehydration and mucus accumulation in cystic fibrosis (CF) patients and in mice with CF-like lung disease. METHODS: We identified several potent ENaC specific antisense oligonucleotides (ASOs) and tested them by inhalation in mouse models of CF-like lung disease. RESULTS: The inhaled ASOs distributed into lung airway epithelial cells and decreased ENaC expression by inducing RNase H1-dependent degradation of the targeted Scnn1a mRNA. Aerosol delivered ENaC ASO down-regulated mucus marker expression and ameliorated goblet cell metaplasia, inflammation, and airway hyper-responsiveness. Lack of systemic activity of ASOs delivered via the aerosol route ensures the safety of this approach. CONCLUSIONS: Our results demonstrate that antisense inhibition of ENaC in airway epithelial cells could be an effective and safe approach for the prevention and reversal of lung symptoms in CF and potentially other inflammatory diseases of the lung.

Reversing Antisense Oligonucleotide Activity with a Sense Oligonucleotide Antidote: Proof of Concept Targeting Prothrombin.

The tissue half-life of second-generation antisense oligonucleotide drugs (ASOs) is generally longer than traditional small molecule therapeutics. Thus, a strategy to reverse the activity of antisense drugs is warranted in certain settings. In this study, we describe a strategy employing the administration of a complementary sense oligonucleotide antidote (SOA). As a model system we have chosen to target the coagulation factor and antithrombotic drug target, prothrombin, to assess the feasibility of this approach. ASO targeting mouse prothrombin specifically suppressed >90% hepatic prothrombin mRNA levels and circulating prothrombin protein in mice. These effects were dose- and time-dependent, and as expected produced predictable increases in anticoagulation activity [prothrombin time/activated partial thromboplastin time (PT/aPTT)]. Treatment with prothrombin SOAs resulted in a dose-dependent reversal of ASO activity, as measured by a return in prothrombin mRNA levels and thrombin activity, and normalization of aPTT and PT. The antithrombotic activity of prothrombin ASOs was demonstrated in a FeCl3-induced thrombosis mouse model, and as predicted for this target, the doses required for antithrombotic activity were also associated with increased bleeding. Treatment with SOA was able to prevent prothrombin ASO-induced bleeding in a dose-dependent manner. These studies demonstrate for the first time the utility of SOAs to selectively and specifically reverse the intracellular effects of an antisense therapy.

Selective depletion of plasma prekallikrein or coagulation factor XII inhibits thrombosis in mice without increased risk of bleeding.

Recent studies indicate that the plasma contact system plays an important role in thrombosis, despite being dispensable for hemostasis. For example, mice deficient in coagulation factor XII (fXII) are protected from arterial thrombosis and cerebral ischemia-reperfusion injury. We demonstrate that selective reduction of prekallikrein (PKK), another member of the contact system, using antisense oligonucleotide (ASO) technology results in an antithrombotic phenotype in mice. The effects of PKK deficiency were compared with those of fXII deficiency produced by specific ASO-mediated reduction of fXII. Mice with reduced PKK had ∼ 3-fold higher plasma levels of fXII, and reduced levels of fXIIa-serpin complexes, consistent with fXII being a substrate for activated PKK in vivo. PKK or fXII deficiency reduced thrombus formation in both arterial and venous thrombosis models, without an apparent effect on hemostasis. The amount of reduction of PKK and fXII required to produce an antithrombotic effect differed between venous and arterial models, suggesting that these factors may regulate thrombus formation by distinct mechanisms. Our results support the concept that fXII and PKK play important and perhaps nonredundant roles in pathogenic thrombus propagation, and highlight a novel, specific and safe pharmaceutical approach to target these contact system proteases.