Suppression of fibrin(ogen)-driven pathologies in disease models through controlled knockdown by lipid nanoparticle delivery of siRNA

Abstract

Fibrinogen plays a pathologic role in multiple diseases. It contributes to thrombosis and modifies inflammatory and immune responses, supported by studies in mice expressing fibrinogen variants with altered function or with a germline fibrinogen deficiency. However, therapeutic strategies to safely and effectively tailor plasma fibrinogen concentration are lacking. Here, we developed a strategy to tune fibrinogen expression by administering lipid nanoparticle (LNP)-encapsulated siRNA targeting the fibrinogen α chain (siFga). Three distinct LNP-siFga reagents reduced both hepatic Fga mRNA and fibrinogen levels in platelets and plasma, with plasma levels decreased to 42%, 16% and 4% of normal within one-week of administration. Using the most potent siFga, circulating fibrinogen was controllably decreased to 32%, 14%, and 5% of baseline with a 0.5, 1, and 2 mg/kg dose, respectively. Whole blood from mice treated with siFga formed clots with significantly decreased clot strength ex vivo, but siFga treatment did not compromise hemostasis following saphenous vein puncture or tail transection. In an endotoxemia model, siFga suppressed the acute phase response and decreased plasma fibrinogen, D-dimer, and proinflammatory cytokine levels. In a sterile peritonitis model, siFga restored normal macrophage migration in plasminogen-deficient mice. Finally, treatment of mice with siFga decreased the metastatic potential of tumour cells in a manner comparable to that observed in fibrinogen-deficient mice. The results indicate that siFga causes robust and controllable depletion of fibrinogen and provide the proof-of-concept that this strategy can modulate the pleiotropic effects of fibrinogen in relevant disease models.

Fibrinogen and Factor XIII in Venous Thrombosis and Thrombus Stability

“…Single-stranded antisense oligonucleotides and small interfering RNA (siRNA) targeting specific fibrinogen chains that reduce circulating fibrinogen without generating fibrin(ogen) degradation products have shown benefit in mouse models of cancer, diet-induced obesity, endotoxemia, peritonitis, and tumor metastasis, without compromising hemostasis. 139,140 Thus, these strategies may also be useful for reducing venous thrombosis in settings of heightened venous thrombosis risk, including constitutively elevated fibrinogen or hyperfibrinogenemia secondary to an inflammatory process.…”

Elimination of fibrin polymer formation or crosslinking, but not fibrinogen deficiency, is protective against diet‐induced obesity and associated pathologies

“…For siFga experiments, male C57BL/6J mice were injected with 2 mg/kg of lipid nanoparticles containing siRNA against luciferase (siLuc) or fibrinogen Aα-chain (siFga) 1 week before the start of the HFD challenge and subsequently every 10-11 days to minimize fluctuations in circulating fibrinogen levels. 17 Mice were fed ad libitum low-fat diet (LFD, 10-13% fat; Laboratory Autoclavable Rodent Diet 5010, LabDiet or D12450, Research Diets) or HFD (60% fat; catalog D12492, Research Diets). The total body weights of mice and the weight of food consumed were determined weekly.…”

“…An unresolved question is whether the α M β 2− binding motif requires fibrin polymerization and crosslinking for the full implementation of fibrin(ogen)‐mediated HFD‐induced obesity and associated pathologies. In this study, the contribution of fibrinogen itself in promoting HFD‐driven obesity was explored using Fibγ 390‐396A mice and mice that are genetically deficient or pharmacologically depleted in fibrinogen (Fib‐ and siRNA targeting fibrinogen [siFga]‐treated, respectively) 17–19 . In addition, to differentiate between distinct fibrin(ogen) molecular forms, we utilized mice expressing fibrinogen locked in the monomeric form (Fib AEK ) and mice deficient in the fibrin crosslinking transglutaminase factor XIII (FXIII–) 20,21 …”

“…[siFga]-treated, respectively). [17][18][19] In addition, to differentiate between distinct fibrin(ogen) molecular forms, we utilized mice expressing fibrinogen locked in the monomeric form (Fib AEK ) and mice deficient in the fibrin crosslinking transglutaminase factor XIII (FXIII-). 20,21…”

Lipid nanoparticles and siRNA targeting plasminogen provide lasting inhibition of fibrinolysis in mouse and dog models of hemophilia A

“…Gene silencing, such as with small interfering RNA (siRNA), has been used in mice to achieve long-lasting control of proteins involved in hemostasis, including fibrinogen ( 10 ), FXI ( 11 ), FXII ( 12 ), and FXIII ( 13 ) and a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13 ( 14 ), and used in clinical trials for bleeding disorders by knocking down antithrombin ( 15 ). Lipid nanoparticles (LNPs) can effectively deliver siRNA to hepatocytes, where plasminogen is synthesized, and have been used in three clinically approved RNA-based therapies ( 16 – 18 ).…”