In vivo reduction of hepatitis B virus antigenemia and viremia by antisense oligonucleotides.

BACKGROUND & AIMS: Current treatment of chronic hepatitis B virus infection (CHB) includes interferon and nucleos(t)ide analogues, which generally do not reduce HBV surface antigen (HBsAg) production, a constellation that is associated with poor prognosis of CHB. Here we evaluated the efficacy of an antisense approach using antisense oligonucleotide (ASO) technology already in clinical use for liver targeted therapy to specifically inhibit HBsAg production and viremia in a preclinical setting. METHODS: A lead ASO was identified and characterized in vitro and subsequently tested for efficacy in vivo and in vitro using HBV transgenic and hydrodynamic transfection mouse and a cell culture HBV infection model, respectively. RESULTS: ASO treatment decreased serum HBsAg levels ⩾2 logs in a dose and time-dependent manner; HBsAg decreased 2 logs in a week and returned to baseline 4 weeks after a single ASO injection. ASO treatment effectively reduced HBsAg in combination with entecavir, while the nucleoside analogue alone did not. ASO treatment has pan-genotypic antiviral activity in the hydrodynamic transfection system. Finally, cccDNA-driven HBV gene expression is ASO sensitive in HBV infected cells in vitro. CONCLUSION: Our results demonstrate in a preclinical setting the efficacy of an antisense approach against HBV by efficiently reducing serum HBsAg (as well as viremia) across different genotypes alone or in combination with standard nucleoside therapy. Since the applied antisense technology is already in clinical use, a lead compound can be rapidly validated in a clinical setting and thus, constitutes a novel therapeutic approach targeting chronic HBV infection.

Antithrombotic effect of antisense factor XI oligonucleotide treatment in primates.

OBJECTIVE: During coagulation, factor IX (FIX) is activated by 2 distinct mechanisms mediated by the active proteases of either FVIIa or FXIa. Both coagulation factors may contribute to thrombosis; FXI, however, plays only a limited role in the arrest of bleeding. Therefore, therapeutic targeting of FXI may produce an antithrombotic effect with relatively low hemostatic risk. APPROACH AND RESULTS: We have reported that reducing FXI levels with FXI antisense oligonucleotides produces antithrombotic activity in mice, and that administration of FXI antisense oligonucleotides to primates decreases circulating FXI levels and activity in a dose-dependent and time-dependent manner. Here, we evaluated the relationship between FXI plasma levels and thrombogenicity in an established baboon model of thrombosis and hemostasis. In previous studies with this model, antibody-induced inhibition of FXI produced potent antithrombotic effects. In the present article, antisense oligonucleotides-mediated reduction of FXI plasma levels by ≥ 50% resulted in a demonstrable and sustained antithrombotic effect without an increased risk of bleeding. CONCLUSIONS: These results indicate that reducing FXI levels using antisense oligonucleotides is a promising alternative to direct FXI inhibition, and that targeting FXI may be potentially safer than conventional antithrombotic therapies that can markedly impair primary hemostasis.

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.

Inhibition of the intrinsic coagulation pathway factor XI by antisense oligonucleotides: a novel antithrombotic strategy with lowered bleeding risk.

Existing anticoagulants effectively inhibit the activity of coagulation factors of the extrinsic and common pathway but have substantial limitations and can cause severe bleeding complications. Here we describe a novel therapeutic approach to thrombosis treatment. We have developed and characterized the efficacy and safety of selective second-generation antisense oligonucleotides (ASOs) targeting coagulation factor XI (FXI), a member of the intrinsic coagulation pathway. Systemic treatment of mice with FXI ASO led to a potent, specific, and dose-dependent reduction of FXI mRNA levels in the liver with corresponding reductions in plasma levels of FXI protein and activity. FXIASO treatment produced potent, dose-dependent antithrombotic activity in various venous and arterial thrombosis models, comparable with warfarin or enoxaparin. However, unlike warfarin or enoxaparin, FXI inhibition did not cause bleeding. Coadministration of FXI ASO with enoxaparin or the antiplatelet drug clopidogrel produced improved antithrombotic activity without increased bleeding. Finally, plasma-derived FXI concentrate was shown to effectively and rapidly reverse the anticoagulant effect of FXI antisense therapy. These results support the concept that inhibition of FXI through antisense therapy might serve as a new and effective strategy for the treatment and prevention of venous thromboembolism with improved specificity and safety.

Inhibition of vascular permeability by antisense-mediated inhibition of plasma kallikrein and coagulation factor 12.

Hereditary angioedema (HAE) is a rare disorder characterized by recurrent, acute, and painful episodes of swelling involving multiple tissues. Deficiency or malfunction of the serine protease inhibitor C1 esterase inhibitor (C1-INH) results in HAE types 1 and 2, respectively, whereas mutations in coagulation factor 12 (f12) have been associated with HAE type 3. C1-INH is the primary inhibitor of multiple plasma cascade pathways known to be altered in HAE patients, including the complement, fibrinolytic, coagulation, and kinin-kallikrein pathways. We have selectively inhibited several components of both the kinin-kallikrein system and the coagulation cascades with potent and selective antisense oligonucleotides (ASOs) to investigate their relative contributions to vascular permeability. We have also developed ASO inhibitors of C1-INH and characterized their effects on vascular permeability in mice as an inducible model of HAE. Our studies demonstrate that ASO-mediated reduction in C1-INH plasma levels results in increased vascular permeability and that inhibition of proteases of the kinin-kallikrein system, either f12 or prekallikrein (PKK) reverse the effects of C1-INH depletion with similar effects on both basal and angiotensin converting enzyme (ACE) inhibitor-induced permeability. In contrast, inhibition of coagulation factors 11 (f11) or 7 (f7) had no effect. These results suggest that the vascular defects observed in C1-INH deficiency are dependent on the kinin-kallikrein system proteases f12 and PKK, and not mediated through the coagulation pathways. In addition, our results highlight a novel therapeutic modality that can potentially be employed prophylactically to prevent attacks in HAE patients.

Mthfd1 is an essential gene in mice and alters biomarkers of impaired one-carbon metabolism.

Cytoplasmic folate-mediated one carbon (1C) metabolism functions to carry and activate single carbons for the de novo synthesis of purines, thymidylate, and for the remethylation of homocysteine to methionine. C1 tetrahydrofolate (THF) synthase, encoded by Mthfd1, is an entry point of 1Cs into folate metabolism through its formyl-THF synthetase (FTHFS) activity that catalyzes the ATP-dependent conversion of formate and THF to 10-formyl-THF. Disruption of FTHFS activity by the insertion of a gene trap vector into the Mthfd1 gene results in embryonic lethality in mice. Mthfd1gt/+ mice demonstrated lower hepatic adenosylmethionine levels, which is consistent with formate serving as a source of 1Cs for cellular methylation reactions. Surprisingly, Mthfd1gt/+ mice exhibited decreased levels of uracil in nuclear DNA, indicating enhanced de novo thymidylate synthesis, and suggesting that serine hydroxymethyltransferase and FTHFS compete for a limiting pool of unsubstituted THF. This study demonstrates the essentiality of the Mthfd1 gene and indicates that formate-derived 1Cs are utilized for de novo purine synthesis and the remethylation of homocysteine in liver. Further, the depletion of cytoplasmic FTHFS activity enhances thymidylate synthesis, affirming the competition between thymidylate synthesis and homocysteine remethylation for THF cofactors.