Preclinical assessment of an optimized AAV-FVIII vector in mice and non-human primates for the treatment of hemophilia A.

Extensive clinical data from liver-mediated gene therapy trials have shown that dose-dependent immune responses against the vector capsid may impair or even preclude transgene expression if not managed successfully with prompt immune suppression. The goal of this preclinical study was to generate an adeno-associated viral (AAV) vector capable of expressing therapeutic levels of B-domain deleted factor VIII (FVIII) at the lowest possible vector dose to minimize the potential Risk of a capsid-mediated immune response in the clinical setting. Here, we describe the studies that identified the investigational agent SPK-8011, currently being evaluated in a phase 1/2 study (NCT03003533) in individuals with hemophilia A. In particular, the potency of our second-generation expression cassettes was evaluated in mice and in non-human primates using two different bioengineered capsids (AAV-Spark100 and AAV-Spark200). At 2 weeks after gene transfer, primates transduced with 2 × 1012 vg/kg AAV-Spark100-FVIII or AAV-Spark200-FVIII expressed FVIII antigen levels of 13% ± 2% and 22% ± 6% of normal, respectively. Collectively, these preclinical results validate the feasibility of lowering the AAV capsid dose for a gene-based therapeutic approach for hemophilia A to a dose level orders of magnitude lower than the first-generation vectors in the clinic.

Coagulation factor V mediates inhibition of tissue factor signaling by activated protein C in mice.

The key effector molecule of the natural protein C pathway, activated protein C (aPC), exerts pleiotropic effects on coagulation, fibrinolysis, and inflammation. Coagulation-independent cell signaling by aPC appears to be the predominant mechanism underlying its highly reproducible therapeutic efficacy in most animal models of injury and infection. In this study, using a mouse model of Staphylococcus aureus sepsis, we demonstrate marked disease stage-specific effects of the anticoagulant and cell signaling functions of aPC. aPC resistance of factor (f)V due to the R506Q Leiden mutation protected against detrimental anticoagulant effects of aPC therapy but also abrogated the anti-inflammatory and mortality-reducing effects of the signaling-selective 5A-aPC variant that has minimal anticoagulant function. We found that procofactor V (cleaved by aPC at R506) and protein S were necessary cofactors for the aPC-mediated inhibition of inflammatory tissue-factor signaling. The anti-inflammatory cofactor function of fV involved the same structural features that govern its cofactor function for the anticoagulant effects of aPC, yet its anti-inflammatory activities did not involve proteolysis of activated coagulation factors Va and VIIIa. These findings reveal a novel biological function and mechanism of the protein C pathway in which protein S and the aPC-cleaved form of fV are cofactors for anti-inflammatory cell signaling by aPC in the context of endotoxemia and infection.

A zymogen-like factor Xa variant corrects the coagulation defect in hemophilia.

Effective therapies are needed to control excessive bleeding in a range of clinical conditions. We improve hemostasis in vivo using a conformationally pliant variant of coagulation factor Xa (FXa(I16L)) rendered partially inactive by a defect in the transition from zymogen to active protease. Using mouse models of hemophilia, we show that FXa(I16L) has a longer half-life than wild-type FXa and does not cause excessive activation of coagulation. Once clotting mechanisms are activated to produce its cofactor FVa, FXa(I16L) is driven to the protease state and restores hemostasis in hemophilic animals upon vascular injury. Moreover, using human or murine analogs, we show that FXa(I16L) is more efficacious than FVIIa, which is used to treat bleeding in hemophilia inhibitor patients. FXa(I16L) may provide an effective strategy to enhance blood clot formation and act as a rapid pan-hemostatic agent for the treatment of bleeding conditions.

Zymogen-like factor Xa variants restore thrombin generation and effectively bypass the intrinsic pathway in vitro.

Inhibitory antibodies to factors VIII or IX represent a serious complication for hemophilia patients. Treatment involves products that bypass the intrinsic pathway and promote thrombin generation. Direct infusion of factor Xa should also restore hemostasis; however, it has a short half-life in plasma and could activate systemic coagulation in an uncontrolled fashion. Here we show that factor Xa mutants with zymogen-like properties (FXa(I16L) and FXa(V17A)) circumvent these limitations. In the absence of factor Va, the FXa variants are poor enzymes for a range of physiological ligands and are resistant to inactivation by antithrombin III and tissue factor pathway inhibitor. Notably, assembly of FXa(I16L) and FXa(V17A) on activated platelets with factor Va to form prothrombinase completely restores biologic activity. In hemophilic plasma, FXa(I16L) and FXa(V17A) have prolonged half-lives compared with wild-type factor Xa (approximately 60 minutes vs approximately 1 minute) and promote robust thrombin generation that bypasses the intrinsic pathway. The variants require factor Va generated in situ for procoagulant function, and cofactor inactivation by the protein C pathway regulates their activity. The efficacy, extended half-life, and mechanism of action suggest that novel zymogen-like forms of factor Xa might prove useful as new therapeutic procoagulants to treat deficiencies upstream of the common pathway.

Recombinant canine B-domain-deleted FVIII exhibits high specific activity and is safe in the canine hemophilia A model.

Production of recombinant B-domain-deleted canine factor VIII (cFVIII-BDD) unexpectedly revealed superior protein yields with 3-fold increased specific activity relative to human FVIII-BDD (hFVIII-BDD). We also determined that activated cFVIII-BDD is more stable than activated hFVIII-BDD. Furthermore, cFVIII-BDD is efficient at inducing hemostasis in human plasma containing FVIII inhibitors. Infusion of cFVIII-BDD in hemophilia A dogs resulted in correction of the disease phenotype with a pharmacokinetic profile similar to clinical experience with hFVIII-BDD. Notably, immune tolerance challenges with cFVIII-BDD in young and adult hemophilia A dogs did not induce the formation of neutralizing or nonneutralizing antibodies to cFVIII. These data establish the framework to quantitatively investigate the efficacy and safety in preclinical studies of novel therapies for hemophilia A.

The conformational switch from the factor X zymogen to protease state mediates exosite expression and prothrombinase assembly.

Zymogens of the chymotrypsin-like serine protease family are converted to the protease state following insertion of a newly formed, highly conserved N terminus. This transition is accompanied by active site formation and ordering of several surface loops in the catalytic domain. Here we show that disruption of this transition in factor X through mutagenesis (FXa(I16L) and FXa(V17A)) not only alters active site function, but also significantly impairs Na(+) and factor Va binding. Active site binding was improved in the presence of high NaCl or with saturating amounts of factor Va membranes, suggesting that allosteric linkage exists between these sites. In line with this, irreversible stabilization of FXa(I16L) with Glu-Gly-Arg-chloromethyl ketone fully rescued FVa binding. Furthermore, the K(m) for prothrombin conversion with the factor Xa variants assembled into prothrombinase was unaltered, whereas the k(cat) was modestly reduced (3- to 4-fold). These findings show that intramolecular activation of factor X following the zymogen to protease transition not only drives catalytic site activation but also contributes to the formation of the Na(+) and factor Va binding sites. This structural plasticity of the catalytic domain plays a key role in the regulation of exosite expression and prothrombinase assembly.

Inhibitory sequences within the B-domain stabilize circulating factor V in an inactive state.

Blood coagulation factor V circulates as a procofactor with little or no procoagulant activity. It is activated to factor Va by thrombin following proteolytic removal of a large central B-domain. Although this reaction is well studied, the mechanism by which bond cleavage and B-domain release facilitate the transition to the active cofactor state has not been defined. Here we show that deletion or substitution of specific B-domain sequences drives the expression of procoagulant function without the need for proteolytic processing. Conversion to the constitutively active cofactor state is related, at least in part, to a cluster of amino acids that is highly basic and well conserved across the vertebrate lineage. Our findings demonstrate that discrete sequences in the B-domain serve to stabilize the inactive procofactor state, with proteolysis primarily functioning to remove these inhibitory constraints. These unexpected results provide new insight into the mechanism of factor V activation.

Role of Hirudin-like factor Va heavy chain sequences in prothrombinase function.

Proexosite I on prothrombin has been implicated in providing a recognition site for factor Va within prothrombinase. To examine whether hirudin-like sequences (659-698) on the cofactor contribute to this interaction, we expressed and purified two-chain FVa derivatives that were intracellularly truncated at the C terminus of the heavy chain: FVa709 (des710-1545), FVa699 (des700-1545), FVa(692 (des693-1545), FVa678 (des679-1545), and FVa658 (des659-1545). We found that FVa709, FVa699, FVa692, and FVa678 exhibited specific clotting activities that were comparable with plasma-derived and recombinant FVa. Additionally, kinetic studies using prothrombin revealed that the Km and kcat values for these derivatives were unaltered. Fluorescent measurements and chromatography studies indicated that FVa709, FVa699, FVa692, and FVa678 bound to FXa membranes and thrombin-agarose in a manner that was comparable with the wild-type cofactors. In contrast, FVa658 had an approximately 1% clotting activity and reduced affinity for FXa membranes (approximately 20-fold) and did not bind to thrombin-agarose. Surprisingly, however, FVa(658) exhibited essentially normal kinetic parameters for prothrombin when the variant was fully saturated with FXa membranes. Overall our results are consistent with the interpretation that any possible binding interactions between prothrombin and the C-terminal region of the FVa heavy chain do not contribute in a detectable way to the enhanced function of prothrombinase.

Characterization of mild coagulation factor VII deficiency: activity and clearance of the Arg315Trp and Arg315Lys variants in the Cys310-Cys329 loop (c170s).

BACKGROUND AND OBJECTIVES: Arginine 315 in factor VII (FVII) belongs to a solvent-exposed loop involved in direct interaction with the co-factor (tissue factor, TF), in transmission of TF-induced effects and potentially in FVIIa inactivation. Natural FVII variants at position 315 provide peculiar models for structure-function studies. DESIGN AND METHODS: We characterized a mild coagulation FVII deficiency associated with reduced FVII activity (26%) and antigen (67%). Mutations were searched by FVII gene sequencing. FVII variants were created by mutagenesis of FVII cDNA and characterized through expression in HEK293 cells followed by functional studies. FVII antigen in media was estimated by immunoassay while FVII activity was assessed by prothrombin-time based and FXa generation assays. FVII variants were injected into mice to investigate their recovery and half-life. One-way ANOVA was used to test statistical significance. RESULTS: The patient was double heterozygous for a novel R315W mutation and for the R304Q substitution (FVII Padua) previously demonstrated to impair TF binding. The recombinant 315W-FVII was normally expressed in medium but showed a markedly reduced coagulant function (52%) and activity towards factor X (FX) in plasma (34%). Moreover, the 315W-FVII showed significantly decreased recovery of the protein (20%) and a slightly shorter half-life (8.6 min) as compared to wt-FVII (50% and 10.7 min). We also studied the conservative R315K change that was responsible for low recovery (20%) and a decreased half-life (7 min) of a FVII variant with virtually normal FVII antigen and activity levels. INTERPRETATION AND CONCLUSIONS: These findings suggest a dual role of R315 for FVII function and clearance, and indicate that substitutions at this position have appreciable effects on human FVII biology, compatible with residual FVII function and thus with mild FVII deficiency.

Removal of B-domain sequences from factor V rather than specific proteolysis underlies the mechanism by which cofactor function is realized.

Factor V, the precursor of factor Va, circulates in plasma with little or no procoagulant activity. Activity is generated following limited proteolysis indicating that the conversion of factor V to factor Va results in appropriate structural changes, which impart cofactor function. We have produced recombinant partial B-domain-truncated derivatives of factor V (FV(des811-1491) and FV(des811-1491) with Arg(709) and Arg(1545) mutated to Gln) to investigate whether discrete proteolysis within the B-domain followed by a conformational transition is responsible for activation. Direct binding fluorescence measurements as well as steady-state kinetic assays were employed to assess the ability of these factor V derivatives to assemble and function in prothrombinase. In contrast to human factor V, single-chain B-domain-truncated factor V bound to FXa membranes with an affinity that was identical to factor Va. Additionally, it was found that, once this modified derivative was assembled in prothrombinase, it functioned in an equivalent manner to factor Va. Taken together these data support the hypothesis that proteolysis within the B-domain of factor V, although necessary, is incidental to the mechanism by which cofactor function is realized. Instead, our results are more consistent with the interpretation that proteolytic activation of factor V simply eliminates steric and/or conformational constraints contributed by the B-domain that otherwise interfere with discrete binding interactions that govern the eventual function of factor Va.

Factor VII mutant V154G models a zymogen-like form of factor VIIa.

Proteolytic cleavage of the peptide bond between Arg(152) and Ile(153) converts the procoagulant protein Factor VII (FVII) to an activated two-chain form (FVIIa). The formation of a salt bridge between Ile(153) and Asp(343) drives the conversion of FVIIa from being zymogen-like to the active form. In the present paper, we describe the novel FVII mutant V154G (Val(154)-->Gly mutation; residue 17 in the chymotrypsin numbering system), found in three FVII-deficient patients, which models a zymogen-like form of FVIIa. Recombinant V154G FVIIa, although normally cleaved, shows markedly reduced activity towards peptidyl substrate and undetectable activity towards macromolecular substrates. Susceptibility of Ile(153) to chemical modification, in either the presence or the absence of tissue factor (TF), suggests that the reduced V154G FVIIa activity is caused by impaired salt-bridge formation, thus resulting in a zymogen-like FVIIa form. The TF-mediated protection from chemical modification of V154A indicated that Gly(154) is responsible for this peculiar feature, and suggests that this region, proximal to the heavy chain N-terminus, is directly involved in the conversion of FVII into FVIIa. V154G FVII was exploited to study the FVII-TF interaction, together with three additional FVII variants that were expressed to serve as models for different FVII forms. The comparison of binding affinities of full-length TF after relipidation in L-alpha-phosphatidylcholine for the zymogen FVII (Arg(152)-->Gln, K (d)=1.04+/-0.27 nM), inactive FVIIa (Ser(344)-->Ala, K (d)=0.27+/-0.06 nM) and a zymogen-like FVIIa (V154G, K (d)=1.15+/-0.16 nM) supports the hypothesis that preferential binding of TF to active FVIIa is insufficient to drive the 10(5)-fold enhancement of FVIIa activity. In addition, the inability of V154G FVIIa to accommodate an inhibitor in the active site, indicating an improperly shaped specificity pocket, would explain the low activity of the zymogen-like form of FVIIa, which is predominant in the absence of TF.

A frequent human coagulation Factor VII mutation (A294V, c152) in loop 140s affects the interaction with activators, tissue factor and substrates.

Activated Factor VII (FVIIa) is a vitamin-K-dependent serine protease that initiates blood clotting after interacting with its cofactor tissue factor (TF). The complex FVIIa-TF is responsible for the activation of Factor IX (FIX) and Factor X (FX), leading ultimately to the formation of a stable fibrin clot. Activated FX (FXa), a product of FVIIa enzymic activity, is also the most efficient activator of zymogen FVII. Interactions of FVII/FVIIa with its activators, cofactor and substrates have been investigated extensively to define contact regions and residues involved in the formation of the complexes. Site-directed mutagenesis and inhibition assays led to the identification of sites removed from the FVIIa active site that influence binding specificity and affinity of the enzyme. In this study we report the characterization of a frequent naturally occurring human FVII mutant, A294V (residue 152 in the chymotrypsin numbering system), located in loop 140s. This region undergoes major rearrangements after FVII activation and is relevant to the development of substrate specificity. FVII A294V shows delayed activation by FXa as well as reduced activity towards peptidyl and macromolecular substrates without impairing the catalytic efficiency of the triad. Also, the interaction of this FVII variant with TF was altered, suggesting that this residue, and more likely loop 140s, plays a pivotal role not only in the recognition of FX by the FVIIa-TF complex, but also in the interaction of FVII with both its activators and cofactor TF.