Titrating haemophilia B phenotypes using siRNA strategy: evidence that antithrombotic activity is separated from bleeding liability

Abstract

Haemophilia A and B are characterised by a life-long bleeding predisposition, and several lines of evidence suggest that risks of atherothrombotic events may also be reduced. Establishing a direct correlation between coagulation factor levels, thrombotic risks and bleeding propensity has long been hampered by an inability to selectively and specifically inhibit coagulation factor levels. Here, the exquisite selectivity of gene silencing combined with a gene knockout (KO) approach was used to define the relative contribution of factor IX (fIX) to thrombosis and primary haemostasis in the rat. Using a lipid nanoparticle (LNP) formulation, we successfully delivered fIX siRNAs to the liver by intravenous administration. The knockdown (KD) of target gene mRNA was achieved rapidly (within 24 hour post-siRNA dosing), sustained (maintained for at least 7 days post dosing) and not associated with changes in mRNA expression levels of other coagulation factors. We found that intermediate levels of liver fIX mRNA silencing (60-95 %) translating into a 50-99 % reduction of plasma fIX activity provided protection from thrombosis without prolonging the cuticle bleeding time. Over 99 % inhibition of fIX activity was required to observe increase in bleeding, a phenotype confirmed in fIX KO rats. These data provide substantial evidence of a participation of fIX in the mechanisms regulating thrombosis prior to those regulating primary haemostasis, therefore highlighting the potential of fIX as a therapeutic target. In addition, hepatic mRNA silencing using LNP-encapsulated siRNAs may represent a promising novel approach for the chronic treatment and prevention of coagulation-dependent thrombotic disorders in humans.

Preclinical and translational evaluation of coagulation factor IXa as a novel therapeutic target

“…2010; Metzger et al. 2015) and performed in the same animal. Male Sprague Dawley rats (350–400 g, Charles River Laboratories, Wilmington, MA, USA) were anesthetized with thiobutabarbital.…”

“…2015). In brief, chemically modified siRNAs were designed against rat fIX (NM 031540) and fX (NM_0143) and synthesized at Merck's oligosynthesis facility (Rahway, NJ, USA).…”

“…2015). In brief, 25  μ L diluted plasma (300X dilution in BSA‐Tris buffer) was incubated with 20  μ L activation reagent containing purified human fXIa (Haematologic Technologies Inc., Essex Junction, VT, USA) for 10 min at 37°C, followed by the addition of 50  μ L of a mixture containing human fX zymogen and activated fVIII, calcium, synthetic phospholipids, and a fXa‐specific substrate SXa‐11.…”

“…Both aPTT and PT were determined by standard methods using TriniCLOT aPTT S (Tcoag, Bray, Ireland) and TriniCLOT PT Excel (Tcoag) on a KC4 Delta coagulation analyzer (Tcoag) (Metzger et al. 2015). Ellagic acid was used as a trigger for aPTT, while 100  μ L tissue factor (TF) + CaCl 2 mix was used to trigger measure of PT.…”

“…We have further confirmed this hypothesis using a short interfering RNA (siRNA) approach in the rat in which a ~50% reduction in fIX plasma activity provided protection from thrombosis with no prolongation of the bleeding time (Metzger et al. 2015). …”