ALBERT

Description: Key Points The binding of administered FVIIa to endogenous EPCR enhances its ability to bypass FVIII or FIX deficiency in vivo. EPCR modulation of function of pharmacologic FVIIa administration may be exploited in protein or gene-based FVIIa therapeutics.

Description: Abstract 201 There is significant interest in developing safe and effective procoagulant hemostatic molecules to treat bleeding disorders. Recently, our laboratory has generated a novel FXa variant (e.g. FXaI16L) with zymogen-like properties that shows promise in bypassing deficiencies upstream of the common pathway. FXaI16L is rendered partially inactive due to a defect in transitioning from zymogen to protease where residue 16 is critical to this process. However, the biological activity of FXaI16L is rescued once its cofactor FVa is made available following coagulation activation. While FXaI16L is effective in correcting the hemophilia phenotype both in vitro and in vivo, the mutation site and general mechanism of action indicates that there is inherent flexibility in this new class of bypassing agent. To this end, we extended the mutational framework at positions 16 and 17 and created a series of zymogenic mutants exhibiting a broad range of activities. We surmised that depending on the amino acid at these positions, the transition from zymogen to protease will be altered to varying degrees. Following an initial screen of ten variants, five were pursued which fell into three categories relative to wild-type (wt)-FXa: “zymogen-like 1” (

Description: Caveolin-1 (CAV1) is a scaffolding protein that is essential for the formation of caveolae membrane domains, functions as a master-regulator of signaling molecules in caveolae, and has major role in the regulation of endothelial nitric oxide synthase (eNOS). Although it is thought that caveolins play some immunomodulatory roles, the actual function of CAV1 with respect to innate immunity in response to bacterial challenge is not clear. The aim of our study was to examine the in vivo role of CAV1 in a mouse model of endotoxemia. CAV1 knock out (ko) and wild-type (WT) mice were intravenously injected with LD80 of E. coli lipopolysaccharide (LPS, 1 mg/kg). Assessment of mortality during 7 days showed that the survival rate of CAV1 ko mice was significantly higher than WT mice (46% vs. 19%). Another group of mice similarly injected with LPS were sacrificed after 2, 8 and 24 hrs, at which times we analyzed multiple parameters of the inflammatory, NO production and coagulation responses in plasma and tissues. Non-challenged CAV1 ko mice have slightly increased numbers of leukocytes comparing to WT mice. After LPS challenge, the amount of circulating leukocytes decreased at 2 hrs in both genotypes, but were significantly lower in CAV1 ko vs. WT mice and correspondingly increased into the tissues (e.g. lung). Interestingly, non-challenged CAV1 ko mice displayed slightly but significantly higher number of neutrophils in the lung than WT mice. After LPS challenge, neutrophils migration increased dramatically at 2 and 24 hrs in WT, but not in CAV1 ko mice. This was paralleled by an increase in myeloperoxidase (a neutrophil product) in WT vs. CAV1 ko mice at 2 hrs and 24 hrs post-challenge. The levels of several cytokines (GM-CSF, IL-1b, IL-5, IL-6, IL-12) were significantly lower in LPS-treated CAV1 ko versus the corresponding WT mice. Only TNF-alpha peaked in CAV1 ko vs. WT mice at 2 hrs post challenge, then decreased at 24 hrs. Several chemokines (KC, IP-10, MCP1, MIG, MIP1a) were transiently upregulated at 2 hrs and diminished at 24 hrs during endotoxemia, but no significant difference between groups was observed. NO generation in response to LPS was evaluated by measuring nitrite/nitrate production in plasma and tissues. While CAV1 ko mice generated higher NO levels during the early stages of sepsis, likely due to increased eNOS function, WT mice displayed 4-fold increase in nitrite/nitrate at 24 hrs, due to a significant upregulation of inducible NOS (iNOS) in tissue leukocytes, as visualized by immunocytochemistry. We observed a temporal correlation between WT mice morbidity and NO generation during the late stages of endotoxemia. Consistent with their increased survival, CAV1 ko mice had lower nitrite/nitrate levels. Non-challenged CAV1 ko mice already have increased tissue factor activity in the lung comparing to WT mice, which further increased at 8 hrs post LPS injection. The resulting procoagulant state was also reflected by elevated levels of thrombin-antithrombin complexes, decreased fibrinogen in plasma, increased fibrin deposition in the lung, increased D-dimmer (peak at 24 hrs after LPS administration) and soluble thrombomodulin plasma levels, as compared with the WT animals. In conclusion, our data reveal that CAV1 ko mice have a more procoagulant but less inflammatory phenotype, which may confer them better survival during endotoxemic challenge. The mechanisms underlying the increased procoagulant and anti-inflammatory functions in CAV1-deficient mice remain to be determined.

Description: The pharmacokinetic challenges of warfarin therapy have led to the development of new oral anticoagulants (NOACs) that directly inhibit coagulation factor Xa (FXa). While these new drugs (including rivaroxaban and apixaban) have many benefits over warfarin, no approved strategy exists to reverse their anticoagulant effects in the event of life-threatening bleeding or emergent need for surgery. The most promising reversal strategy in clinical development is a catalytically inactive form of FXa (Gla-domainless FXaS195A, GD-FXaS195A) that binds the NOAC with high affinity to relieve inhibition of endogenous FXa. In principle, removing the inhibitor through molecular engagement is attractive, especially when administered before bleeding begins (e.g. pre-surgery), and there is in vivo evidence to support the efficacy of GD-FXaS195A as a pre-injury antidote for direct FXa inhibitors. However, since GD-FXaS195A is a scavenger of the inhibitor and not a pro-hemostatic agent, GD-FXaS195A may not be effective if given after a bleeding episode has begun. Moreover, its mechanism of action necessitates a 1:1 ratio of GD-FXaS195A to neutralize the inhibitor. Thus, high doses (hundreds of milligrams of protein) will likely be required in humans. We hypothesized that a pro-hemostatic bypassing agent might be able to reverse the effects of direct FXa inhibitors no matter when it is administered, and with high potency. To evaluate this, we used a variant of FXa (FXaI16L) that is more zymogen-like than wild-type (wt)-FXa. This "zymogen-like" variant has lower catalytic activity in in vitro assays compared to wt-FXa due to impaired active site maturation. It is also resistant to active site inhibitors including plasma protease inhibitors, resulting in an extension of its plasma half-life (〉30 minutes vs ~1 minute for wt-FXa). Importantly, its activity is rescued upon binding to its cofactor FVa in vivo, and we have previously shown that FXaI16L bypasses the intrinsic pathway defect in hemophilic mice. Here we evaluated whether FXaI16L might also be able to bypass direct FXa inhibitors using in vitro thrombin generation assays (TGAs) and two in vivo injury models in mice, and directly compared FXaI16L to GD-FXaS195A. In TGA experiments, 500 nM rivaroxaban decreased peak thrombin generation to 23% of that observed in normal human plasma (NHP). This decreased thrombin generation could be reversed by the addition of 3 nM human (h)FXaI16L which normalized peak thrombin generation. In comparative studies, hFXaI16L was 300-fold more potent than GD-FXaS195A in TGA assays. These data highlight that differences in mechanism of action (pro-hemostatic vs. scavenger) will have a huge impact on amounts of protein needed to revive thrombin generation. This was further reflected in in vivo studies. In invivo experiments with wild-type mice using 7.5% FeCl3 to induce carotid artery thrombosis, 1 mg/kg rivaroxaban prevented formation of occlusive thrombi. 30 minutes after the initial FeCl3 injury to rivaroxaban-treated mice, infusion of 0.25 mg/kg murine (m)FXaI16L normalized the phenotype, causing rapid occlusion at the injury site within 5 minutes. In contrast, we did not observe carotid artery occlusion with administration of up to 25 mg/kg GD-FXaS195A 30 minutes after the injury to rivaroxaban-treated mice (Fig. 1). When both rivaroxaban and the reversal agent were given prior to the injury, both mFXaI16L (0.5 mg/kg) and GD-FXaS195A (25 mg/kg) were effective at reversing the inhibition. Furthermore, using intravital microscopy to examine thrombus formation at the site of laser injury to mouse cremasteric arterioles, we found that 1 mg/kg rivaroxaban completely abrogated fibrin deposition and substantially reduced platelet accumulation. Treatment with1 mg/kg mFXaI16L substantially increased platelet and fibrin deposition at the site of laser injury. Figure 1 Carotid occlusion time when the reversal agent was infused 30 minutes after the FeCl3 injury. Figure 1. Carotid occlusion time when the reversal agent was infused 30 minutes after the FeCl3 injury. Taken together, these data provide strong support for the in vivo efficacy and potency of FXaI16L as a potential pro-hemostatic bypass agent to reverse direct FXa inhibitors. The data also illustrate that antidotes like GD-FXaS195A may not be as effective as pro-hemostatic agents like FXaI16L following an injury, and we predict that effective reversal of NOACs will require a nuanced approach that uses an antidote or a bypassing agent depending on the clinical scenario. Disclosures Jasuja: Pfizer: Employment. Patel-Hett:Pfizer: Employment. Fruebis:Pfizer: Employment. Pittman:Pfizer: Employment. Camire:Pfizer: Consultancy, Patents & Royalties, Research Funding.

Description: Abstract 18 High-dose human activated Factor VII (FVIIa) use is widespread in hemophilic patients with anti Factor VIII or Factor IX antibodies as well as in off-label applications. In addition to tissue factor, the endothelial protein C receptor (EPCR) also binds human FVIIa. However, the physiological consequences of this interaction on the potential hemostatic effects following bolus administration of FVIIa are still unclear. To investigate this in a mouse model, we decided to study the interaction of murine FVIIa (mFVIIa) with murine EPCR (mEPCR) in vitro and, subsequently, in vivo. We have previously shown, either in solution (with murine soluble EPCR) or on cells expressing full-length EPCR, that mFVIIa has a very low affinity for murine EPCR (Kd 〉 8μM), in contrast to human FVIIa binding to human EPCR. Therefore, to use the mouse as a model to study the FVIIa-EPCR interaction, we engineered mEPCR binding capacity into mFVIIa by partial substitution of its Gla domain, using the murine Protein C Gla domain as a donor. Combined modifications of 3 residues in mFVIIa (L4F, L8M and W9R; FMR-mFVIIa) were sufficient to confer mEPCR binding (Kd ≈ 200 nM, in presence of 1.6 mM Ca2+ and 0.6 mM Mg2+), without impairing the activity of the molecule as measured by a clotting assay. Here, we extend the characterization of FMR-mFVIIa in vitro and in vivo. First, we monitored the affinity of FMR-mFVIIa or wildtype mFVIIa (WT-mFVIIa) for its natural cofactor, murine tissue factor in the context of a cell membrane. For this, we generated CHO-K1 cells stably expressing full-length mTF (CHO-K1-mTF). FMR- or WT-mFVIIa was incubated at 4 degrees C (in presence of 1.6 mM Ca2+ and 0.6 mM Mg2+) on such cells at increasing concentration and, following quantification of the bound fraction, we observed no difference between FMR- and WT-mFVIIa in affinity for mTF (18 ± 13 nM and 17 ± 2 nM, respectively). To begin defining the role of EPCR-FVIIa interaction in vivo, we injected WT-mFVIIa or FMR-mFVIIa (500μg/kg) into C57BL6 wildtype mice (n=5 per protein per timepoint) via tail vein and monitored plasmatic concentration at different timepoints. The decrease in plasmatic levels over time followed a biphasic pattern. At 5 and 15 minutes post injection, plasmatic concentration of FMR-mFVIIa was significantly lower than WT-mFVIIa (41 ± 9% [FMR-mFVIIa] vs. 66 ± 7% [WT-mFVIIa] of the initial dose at 5 min, p=0.001; 18.5 ± 2.8% [FMR-mFVIIa] vs. 40.5 ± 9.0% [WT-mFVIIa] of the initial dose at 15 min, p=003). No differences were observed at later timepoints (up to 2 hours post protein infusion). Moreover, there were no changes in either platelet counts or hematocrit over the period of observation. Next, we wanted to confirm that the differences in recovery between the infused proteins were the result of mEPCR binding. For this, we infused an EPCR-blocking (RCR-252) or isotype control antibody (50μg/mouse) prior to administration of FMR- or WT-mFVIIa. We assessed plasmatic concentration at 5 min post protein infusion. In accordance with our previous data, mice that received isotype control IgG showed reduced recovery for the FMR-mFVIIa chimera vs. WT-mFVIIa (p=0.001). In contrast, antibody blocking of mEPCR prior to protein infusion increased the recovery of FMR-mFVIIa to that observed for WT-mFVIIa. These data suggest that the reduced recovery observed by bolus administration of FMR-mFVIIa vs. WT-mFVIIa was attributable to the mEPCR binding capacity of FMR-mFVIIa. In conclusion, we have now characterized a mFVIIa chimeric molecule indistinguishable from WT-mFVIIa in terms of mTF binding and clotting activity, but bearing the capacity to interact with mEPCR in vitro and, more importantly, in vivo. These features mimic those found in human FVIIa, thereby allowing the study of EPCR-dependent mechanisms in the clearance and/or biodistribution of FVII/FVIIa in vivo. Our observations suggest, for the first time in a homologous system, that EPCR-binding capacity has a specific negative effect on the recovery of the mFVIIa chimera. This molecule can now be utilized in the context of bolus protease administration in hemophilic mice following injury, to test any potential hemostatic effects from a FVIIa-EPCR interaction in vivo. This may provide additional insight into the mechanism of action of high-dose FVIIa administration in hemophilia. Disclosures: Pavani: Bayer: Research Funding. Margaritis:Novo Nordisk A/S: Research Funding; Bayer: Research Funding.

Description: Abstract LBA-5 Inherited hematologic disorders have the potential to be effectively treated by gene therapy, with recent successes reported for several genetic disorders using viral vector-mediated gene transfer (ADA-SCID, NEJM 2009; β-thalassemia, Nature 2010). However, these trials and others illustrate some of the disadvantages and risks of using viral vector-based gene addition strategies, including loss of endogenous gene regulation and random insertion leading to potential for insertional mutagenesis. An alternative approach is gene correction, where in situ correction of a gene mutation allows endogenous gene regulation and decreases risks related to random integration. Gene correction is based on gene targeting, the therapeutic utility of which has historically been limited to mouse embryonic stem cells due to low homologous recombination rates in other cell types. However, a recently developed class of fusion proteins, zinc finger nucleases (ZFNs), have been shown to increase targeting efficiency 2–3 logs by inducing site-specific DNA double strand breaks at the intended targeting site. ZFNs have permitted high efficiency therapeutic gene targeting in a variety of cultured cells previously thought intractable to these processes, but ZFN-mediated gene correction has yet to be successfully achieved in vivo in an animal model of disease. Here we show ZFN-mediated therapeutic gene targeting of a mutated F9 gene in vivo, resulting in phenotypic correction of a mouse model of hemophilia B (HB). We first generated ZFNs targeting intron 1 of the human F9 gene (F9 ZFNs). We hypothesized the F9 ZFNs would mediate insertion of a wild-type F9 exons 2–8 minigene into intron 1 via gene targeting, thus bypassing the 95% of F9 mutations that occur in exons 2–8. We next generated a humanized HB mouse model with a deletion of the mouse F9 gene and knock-in (at the ROSA 26 locus) of a catalytic domain-deleted human F9 mini-gene (hF9mut) transgene. Adeno-associated viral (AAV) vector delivery of the F9 ZFNs to hF9mut mouse liver resulted in cleavage of the intron 1 target site in 45% of hepatocytes. We then generated an AAV donor vector containing a w.t. exons 2–8 insert flanked by arms of homology. Co-delivery of the AAV-ZFN and AAV-donor vectors to neonatal hF9mut mice (n=16) resulted in circulating F.IX levels of 120–350 ng/mL (2-7% of normal), whereas mice receiving AAV-ZFN alone (n=17) or AAV-mock & AAV-donor (n=15) had no detectable F.IX expression (detection limit 15 ng/mL), or

Description: 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 gene-transfer 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 addition, we confirmed the therapeutic efficacy of FIX variants in mice with inhibitory Abs against FVIII. Further, mice tolerant to wild-type human FIX did not develop immune responses against the protein variants. Our results therefore indicate the feasibility of using variants of FIX to bypass FVIII as a novel treatment approach in hemophilia with and without neutralizing FVIII Abs.