FIX-Triple, a gain-of-function factor IX variant, improves haemostasis in mouse models without increased risk of thrombosis

Abstract

Engineered recombinant factor IX (FIX) with augmented clotting activity may prove useful for replacement therapy, but it has not been studied for risk of thrombosis. We used three mouse models to evaluate thrombosis risk associated with the FIX variant FIX-Triple, which has a 13-fold higher specific activity than wild-type FIX (FIX-WT). Protein infusion of FIX-Triple into haemophilia B mice was not thrombogenic, even at a dose of 13-fold higher than FIX-WT. Gene knock-in to generate mice that constitutively produce FIX-WT or FIX-Triple protein revealed that all mice expressed equal antigen levels. FIX-Triple knock-in mice that exhibited 10-fold higher FIX clotting activity did not show hypercoagulation. Adeno-associated viral (AAV) delivery of the FIX gene into mice was used to mimic gene therapy. Haemophilia B and inbred C57Bl/6 mice injected with different doses of virus particles carrying FIX-WT or FIX-Triple and expressing up to a nearly 13-fold excess (1289% of normal) of FIX clotting activity did not show increased risk of thrombosis compared with untreated wild-type mice in a normal haemostatic state. When challenged with ferric chloride (FeCl3), the mesenteric venules of AAV-treated C57Bl/6 mice that gave a nearly five-fold excess (474%) of FIX clotting activity were not thrombotic; however, thrombosis became obvious in FeCl3-challenged mice expressing extremely high FIX clotting activities (976-1289%) achieved by AAV delivery of FIX-Triple. These studies suggest that FIX-Triple is not thrombogenic at therapeutic levels and is a potential therapeutic substitute for FIX-WT.

Gene therapy for hemophilia

“…More specific to F.IX, though, is the discovery of mutants with increased clotting activity. Alanine substitution mutation resulted in the artificial generation of F.IX-triple, which has ~10-fold greater specific activity than wild-type F.IX (211, 212). Additionally, a naturally occurring F.IX mutation (R338L), termed F.IX Padua, was discovered that has 5-10-fold higher activity (42, 213-215).…”

Animal Models of Hemophilia

“…144 This latter knockout model has in fact been used to generate a number of additional strains each of which expresses a single copy F9 gene from the “socket” located in the endogenous single copy mouse F9 gene locus. These strains include: mouse FIX expressing a mutation that alters the FIX Gla domain interaction with endothelium (K5AFIX) 145 defective human FIX having a missense mutation at a critical arginine in the catalytic domain (R333QFIX) 146; 147 defective human FIX having a nonsense mutation in the Gla domain that results in an early stop mutation (R29XFIX) 148 human FIX wild type (FIX-WT mouse) 147 human FIX carrying three mutations each of which increases the specific activity of the protein (“FIX Triple”). 147 …”

“…defective human FIX having a missense mutation at a critical arginine in the catalytic domain (R333QFIX) 146; 147 …”

“…A bioengineered FIX mouse has recently been reported by Shu-Wha Lin’s group that has a gene knock-in of a F9 gene incorporating an Arg338Ala (previously characterized) 249192; 250 and two additional gain of function mutations. 147; 251 Mice with endogenous expression of this increased specific activity “FIX Triple” have a hemostatic phenotype that is not complicated by inappropriate activation of coagulation. Incorporating similar gain of function FIX variants into AAV vectors may permit improved correction of hemophilia while further decreasing vector capsid load.…”

The efficacy and the risk of immunogenicity of FIX Padua (R338L) in hemophilia B dogs treated by AAV muscle gene therapy

“…In an early study, we demonstrated that replacement of arginine 338 by alanine (R338A) was associated with an ϳ 3-fold increase in the protein specific activity in murine models of HB receiving AAV-FIX-R338A 9 as later confirmed in other models. 10,11 Recently, we described a naturally occurring gain-of-function mutation in humans characterized by leucine at position 338 (R338L), which exhibits normal antigen levels, but an ϳ 8-fold higher specific activity. 12 Notably, the arginine at position 338 in FIX is conserved among mammals, and this region of the enzyme appears to be part of the substrate exosite for factor X.…”