Engineered factor IX variants bypass FVIII and correct hemophilia A phenotype in mice

Abstract

The complex of the serine protease factor IX (FIX) and its cofactor, factor VIII (FVIII), is crucial for propagation of the intrinsic coagulation cascade. Absence of either factor leads to hemophilia, a disabling disorder marked by excessive hemorrhage after minor trauma. FVIII is the more commonly affected protein, either by X-chromosomal gene mutations or in autoimmune-mediated acquired hemophilia. Whereas substitution of FVIII is the mainstay of hemophilia A therapy, treatment of patients with inhibitory Abs remains challenging. In the present study, we report the development of FIX variants that can propagate the intrinsic coagulation cascade in the absence of FVIII. FIX variants were expressed in FVIII-knockout (FVIII-KO) mice using a nonviral genetransfer system. Expression of the variants shortened clotting times, reduced blood loss after tail-clip assay, and reinstalled clot formation, as tested by in vivo imaging of laser-induced vessel injury. In IntroductionThe intrinsic coagulation cascade is a tightly regulated proteaseand cofactor-dependent amplification system that ensures the formation of stable clots after injury. 1 Within this system, deficiencies of the coagulation cofactor, factor VIII (FVIII), or the corresponding coagulation protease, factor IX (FIX), lead to the X-chromosomal inherited bleeding disorders hemophilia A and hemophilia B, respectively. Hemophilia A occurs approximately in 1 of 5000 newborn boys, whereas hemophilia B is less common. 2 Untreated, hemophilia presents with spontaneous bleeding preferentially into large joints and skeletal muscle, and internal and intracranial bleedings can also occur. Substitution of deficient coagulation factors by intravenous infusion of plasma-derived or recombinant coagulation factor concentrates is the therapy of choice. In the last decades, treatment has evolved from so-called on-demand treatment for acute injury/hemorrhage to a secondary preventative approach with regular prophylactic infusions. Prophylactic treatment, securing plasma levels above 1% of normal, already prevents the major long-term consequences of the disease: joint damage and muscular atrophy. 3,4 A major obstacle for protein substitution therapy is the occurrence of neutralizing Abs directed against the FVIII protein. This can either be a result of an immune response after exogenous protein exposure 5 or may appear in adult patients as a spontaneous auto-immune event. 6 In these cases, FVIII infusion is often ineffective and so-called bypassing agents are used. These agents consist of constitutively activated proteases such as activated factor VII (FVIIa), and promote clot formation directly without restoring the intrinsic amplification loop. Although these therapeutics are efficient at stopping acute bleeding, limitations include the relatively short half-lives of activated proteases in the circulation and potential vascular risks in long-term treatment. 7,8 In the present study, we report on the generation of FIX variants with FVIII-independent clotting activity that we...

Rate-limiting roles of the tenase complex of factors VIII and IX in platelet procoagulant activity and formation of platelet-fibrin thrombi under flow

“…45 Interestingly, engineered FIX variants that stimulate the coagulation process independently of FVIII appeared to reduce bleeding and normalize in vivo thrombus formation in hemophilic mice lacking FVIII. 46 This latter observation is compatible with a primary role of FVIII in (soluble?) FIX activation, and the concept that thrombus-localized active FIX is sufficient for FX activation and ensuing thrombin generation.…”

Engineered Factor VII, Factor IX, and Factor X Variants for Hemophilia Gene Therapy

“…The variants T and ITV did not exhibit any signs for pre-activation of the FIX zymogen [58]. Using a nonviral gene transfer system [60] expression of the variants T and ITV in FVIII knockout mice resulted in shortening of clotting times, reduced blood loss and recovery of clot formation in the microvasculature as observed by imaging of laser induced vessel injury [58]. Correction of the bleeding phenotype was additionally demonstrated in presence of FVIII inhibitory antibodies [58].…”

“…Furthermore, by combining K265T with two further mutations (V181I and I383V) in the same study the authors obtained a FIX variant (ITV) with 15.6% activity in absence of FVIII at physiological FIX levels. The variants T and ITV did not exhibit any signs for pre-activation of the FIX zymogen [58]. Using a nonviral gene transfer system [60] expression of the variants T and ITV in FVIII knockout mice resulted in shortening of clotting times, reduced blood loss and recovery of clot formation in the microvasculature as observed by imaging of laser induced vessel injury [58].…”

“…Using a nonviral gene transfer system [60] expression of the variants T and ITV in FVIII knockout mice resulted in shortening of clotting times, reduced blood loss and recovery of clot formation in the microvasculature as observed by imaging of laser induced vessel injury [58]. Correction of the bleeding phenotype was additionally demonstrated in presence of FVIII inhibitory antibodies [58]. Therefore the usage of FIX variants with independent FVIIII activity might provide an innovative FVIII bypassing therapy for patients with neutralizing anti-FVIII antibodies.…”

“…A FIXa variant with triple mutation (Y259F/K265T/Y345T) exhibited several 1000-fold increases in amidolytic activity toward synthetic peptide substrates and altered substrate specificity [56]. Based on these outcomes [56,57], Milanov et al explored whether introduction of the mutations Y259F, K265T, and Y345T into the full length FIX protein would give rise to cofactor independent activity under normal physiological conditions by determining its clotting activities in human FIX-, FVIII-and FX-deficient plasma [58]. As a result, the triple variant exposed almost the same FIX clotting activity as wt-FIX and no shift toward FX substrate affinity was observed.…”