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Background Prophylactic replacement therapy in hemophilia A (HA) patients does not adequately prevent bleeds and arthropathic complications. A more refined understanding of the relationship between coagulation factor VIII (FVIII) levels and bleeding risk during protein prophylaxis, or with gene therapy, is needed to improve patient care. Objectives Investigate this relationship in the HA rat, a model exhibiting spontaneous bleeds and development of arthropathy similar to HA patients. Methods Human B domain-deleted FVIII was delivered to HA rats via adeno-associated virus (AAV)-mediated gene transfer or multiple intravenous protein injections. Results and conclusions After 12 weeks of observation, both approaches significantly reduced bleeds per animal and increased the proportion of bleed-free animals compared with controls (43% vs 0%, respectively [AAV]; 75% vs 8%, respectively [injection]). Both approaches resulted in an anti-FVIII inhibitory response in 20% to 37% of treated animals, similar to HA patients. Inhibitory antibodies were refractory to clinical improvement (reduction of bleeds) only in the AAV-based prophylaxis. An integrated model-based analysis of data on FVIII exposure and bleeding events was performed. This predicted the bleeding risk at any given circulating FVIII activity. Specifically, 4.8 or 10 IU/dL FVIII (0.048 and 0.1 IU/mL, respectively) were predicted to reduce bleeding risk by 90% or 95%, respectively, compared with untreated controls. Our data establish the utility of the HA rat model in FVIII prophylaxis studies and describe how FVIII activity affects bleeding risk in this setting. These enable further studies on FVIII prophylaxis focusing on disease complications for an optimized treatment of HA patients.

Introduction Monitoring of clinical effectiveness of bypassing agents in haemophilia patients is hampered by the lack of validated laboratory assays. Thromboelastography (TEG) and rotational thromboelastometry (ROTEM) have been evaluated for predicting clinical effectiveness of bypassing agents, however, with limited success. Aim Application of a longitudinal model-based approach may allow for a quantitative characterization of the link between ROTEM parameters and the probability of bleeding events. Methods We analyse longitudinal data from haemophilia A rats receiving gene-based FVIIa prophylaxis in terms of total circulatory levels of FVII/FVIIa, clotting time (CT) measured using ROTEM and the probability of bleeding events. Results Using population pharmacokinetic-pharmacodynamic (PKPD) modelling, a PK-CT-repeated time-to-event (RTTE) model was developed composed of three submodels (a) a FVII/FVIIa PK model, (b) a PK-CT model describing the relationship between predicted FVIIa expression and CT and (c) a RTTE model describing the probability of bleeding events as a function of CT. The developed PK-CT-RTTE model accurately described the vector dose-dependent plasma concentration-time profile of total FVII/FVIIa and the exposure-response relationship between AAV-derived FVIIa expression and CT. Importantly, the developed model accurately described the occurrence of bleeding events over time in a quantitative manner, revealing a linear relationship between predicted change from baseline CT and the probability of bleeding events. Conclusion Using PK-CT-RTTE modelling, we demonstrated that ROTEM parameters can accurately predict the probability of bleeding events in a translational animal model of haemophilia A.

Methamphetamine (METH) continues to be among the most addictive and abused drugs in the United States. Unfortunately, there are currently no Food and Drug Administration–approved pharmacological treatments for METH-use disorder. We have previously explored the use of adeno-associated viral (AAV)-mediated gene transfer of an anti-METH monoclonal antibody. Here, we advance our approach by generating a novel anti-METH single-chain variable fragment (scFv)-Fc fusion construct (termed 7F9-Fc) packaged into AAV serotype 8 vector (called AAV-scFv-Fc) and tested in vivo and ex vivo. A range of doses [1 × 10(10), 1 × 10(11), and 1 × 10(12) vector copies (vcs)/mouse] were administered to mice, eliciting a dose-dependent expression of 7F9-Fc in serum with peak circulating concentrations of 48, 1785, and 3831 µg/ml, respectively. Expressed 7F9-Fc exhibited high-affinity METH binding, IC(50) = 17 nM. Between days 21 and 35 after vector administration, at both 1 × 10(11) vc/mouse and 1 × 10(12) vc/mouse doses, the AAV-7F9-Fc gene therapy significantly decreased the potency of METH in locomotor assays. On day 116 post–AAV administration, mice expressing 7F9-Fc sequestered over 2.5 times more METH in the serum than vehicle-treated mice, and METH concentrations in the brain were reduced by 1.2 times the value for vehicle mice. These data suggest that an AAV-delivered anti-METH Fc fusion antibody could be used to persistently reduce concentrations of METH in the central nervous system. SIGNIFICANCE STATEMENT: In this manuscript, we describe the testing of a novel antimethamphetamine (METH) single-chain variable fragment–Fc fusion protein delivered in mice using gene therapy. The results suggest that the gene therapy delivery system can lead to the production of significant antibody concentrations that mitigate METH’s psychostimulant effects in mice over an extended time period.

The development of neutralizing antibodies (inhibitors) to factor VIII (FVIII) remains the most challenging complication of protein-based replacement therapy in hemophilia A (HA). Elevated levels of the B cell survival cytokine B cell activating factor (BAFF) are associated with inhibitors in HA patients and BAFF levels decrease with successful immune tolerance induction (Doshi BS et al, J Clin Invest 2021). In HA mice, localization of labeled FVIII to the spleen and subsequent depletion of splenic leukocytes has been shown to reduce and/or temporarily prevent FVIII inhibitors. In contrast, transient depletion of BAFF prevented inhibitors upfront and promoted long-term tolerance to FVIII in HA mice. Given BAFF's ability to modulate B cell pools in secondary lymphoid organs, here we compared the splenic versus extra-splenic role of BAFF in regulating the FVIII immune response in order to localize its mechanism of action. As BAFF could regulate survival of innate-like splenic-resident marginal zone B cells, initial experiments compared depletion of BAFF or marginal zone B cells in HA mice and showed more complete inhibitor prevention with BAFF depletion (median 0 vs 16.9 Bethesda Units [BU]/ml, respectively, p < 0.01). These data supported our hypothesis that BAFF's regulatory effect is not solely reliant on its modulation of the splenic-resident marginal zone B cell compartment in HA mice. To further understand the extra-splenic role of BAFF in FVIII immunogenicity, wild-type (WT) or severe HA mice (with F8 exon 16 knockout) on the C57Bl/6 background, were treated according to three groups (n=9-11/group): 1) sham controls, 2) splenectomy alone, and 3) anti-BAFF monoclonal antibody (2mg/kg IP once) 4 days prior to splenectomy ("combined therapy"). Following splenectomy, mice were injected with recombinant full length human FVIII at 80 IU/kg weekly for six weeks. Bethesda titers were measured one week following the final immunization. In order to provide hemostasis in HA mice without FVIII exposure prior to splenectomy, mice were given liver-directed gene therapy with AAV8-mFVIIa at 5e11 vg/mouse two weeks prior to splenectomy. Circulating mFVIIa levels of 5.8 ± 2.4 ug/ml at day 10 were observed. These levels normalized hemostasis allowing successful splenectomy in 85% of animals, which is similar to our experience in WT mice. In the combined therapy group, only a single HA mouse developed a high titer inhibitor (7.4 BU/ml) and no WT mice developed high titer inhibitors. As the presence of endogenous FVIII antigen in WT mice does not seem to significantly alter BAFF's role in preventing the alloantibody response to xenoantigen (human FVIII protein), data for WT and HA mice were combined for analysis. Although splenectomy decreased inhibitors titers compared to sham controls (23.1 vs 78.7 BU/ml), the addition of anti-BAFF antibody therapy to splenectomy significantly reduced the risk of high titer inhibitors (RR 0.15, 95% CI 0.02-0.66, p < 0.01). The median Bethesda titer in HA and WT splenectomized mice was 23.1 (0-139.4) versus 0 (0-7.4) with addition of BAFF antibody therapy. Of note, inhibitor prevention with combination splenectomy and anti-BAFF antibody therapy seen here is similar to our prior data using just anti-BAFF antibody monotherapy for FVIII inhibitor prevention in HA mice (RR 0.23, 95% CI 0.08-0.57) suggesting that splenectomy does not significantly improve FVIII inhibitor prevention in the setting of BAFF inhibition. Our data, using a challenging HA mouse model, suggest that anti-BAFF therapy may prevent FVIII inhibitors by inhibiting BAFF's function in non-splenic lymphoid compartments. As certain B cell subtypes, including marginal zone B cells, reside in non-splenic compartments in humans, our model mimics the anticipated human immune response to FVIII. Ongoing investigations into the B and T cell compartments in lymph nodes and bone marrow will provide further insights into the location of BAFF's regulatory role in the FVIII immune response and may provide insights into potential alternative pathways in the initial FVIII immune response. Disclosures Doshi: Janssen: Consultancy; Spark Therapeutics: Speakers Bureau.

Methamphetamine (METH) continues to be amongst the most addictive and abused drugs in the US. Unfortunately, there are currently no FDA approved pharmacological treatments for METH substance abuse disorder. As an alternative approach, we have previously explored the use of Adeno-associated viral (AAV) mediated gene transfer of an anti-METH monoclonal antibody. Here, we advance our approach by generating a novel anti-METH scFv-Fc fusion construct (7F9-Fc), packaged into AAV serotype 8 vector (called AAV-scFv-Fc), and tested in vivo and ex vivo. A range of doses (1 × 1010. 1 × 1011, and 1 × 1012 vector copies(vc)/mouse) were administered to mice, which exhibited a dose-dependent expression of 7F9-Fc in serum with peak circulating concentrations of 48, 1785, and 3,831 μg/ml. The dose of 1 × 1012 vc/mouse was further tested in METH locomotor and biodistribution studies to determine the efficacy of the antibody protection. Expressed 7F9-Fc exhibited high affinity binding, 17 nM, to METH. Between days 21 and 35 after vector administration, the 7F9-Fc gene therapy significantly reduced the effects of METH in locomotor assays following administration of moderate and high doses of subcutaneous METH, 3.1 and 9.4 mg/kg respectively. On day 116 post-AAV administration, mice expressing 7F9-Fc sequestered over 2.5 times more METH into the serum than vehicle mice, and METH concentrations in the brain were reduced by 1.2 times compared to vehicle mice. Taken together, these data suggest that a AAV-delivered anti-METH Fc fusion antibody could be a design for persistently reducing concentrations of METH in the CNS.Significance StatementIn this manuscript, we describe the use of a novel anti-METH scFv-Fc fusion protein delivered in mice using gene therapy. The results suggest that the gene therapy delivery system can lead to the production of enough antibody to mitigate METH’s psychostimulant effects in mice over an extended time period.

Coagulation and inflammation are interconnected, suggesting that coagulation plays a key role in the inflammatory response to pathogens. A phenome-wide association study (PheWAS) was used to identify clinical phenotypes of patients with a polymorphism in coagulation factor X. Patients with this single nucleotide polymorphism (SNP) were more likely to be hospitalized with hemostatic and infection-related disorders, suggesting that factor X contributes to the immune response to infection. To investigate this, we modeled infections by human pathogens in a mouse model of factor X deficiency. Factor X-deficient mice were protected from systemic Acinetobacter baumannii infection, suggesting that factor X plays a role in the immune response to A. baumannii. Factor X deficiency was associated with reduced cytokine and chemokine production and alterations in immune cell population during infection: factor X-deficient mice demonstrated increased abundance of neutrophils, macrophages, and effector T cells. Together, these results suggest that factor X activity is associated with an inefficient immune response and contributes to the pathology of A. baumannii infection.

Factor VII (FVII) deficiency is a rare autosomal recessive bleeding disorder treated by infusion of fresh-frozen plasma, plasma-derived FVII concentrates and low-dose recombinant activated FVII. Clinical data suggest that a mild elevation of plasma FVII levels (10% normal) results in improved hemostasis. Research dogs with a G96E missense FVII mutation (FVII-G96E) have1% FVII activity. By western blot, we show that they have undetectable plasmatic antigen, thus representing the most prevalent type of human FVII deficiency (low antigen/activity). In these dogs, we determine the feasibility of a gene therapy approach using liver-directed, adeno-associated viral (AAV) serotype 8 vector delivery of a canine FVII (cFVII) zymogen transgene. FVII-G96E dogs received escalating AAV doses (2E11 to 4.95E13 vector genomes [vg] per kg). Clinically therapeutic expression (15% normal) was attained with as low as 6E11 vg/kg of AAV and has been stable for1 year (ongoing) without antibody formation to the cFVII transgene. Sustained and supraphysiological expression of 770% normal was observed using 4.95E13 vg/kg of AAV (2.6 years, ongoing). No evidence of pathological activation of coagulation or detrimental animal physiology was observed as platelet counts, d-dimer, fibrinogen levels, and serum chemistries remained normal in all dogs (cumulative 6.4 years). We observed a transient and noninhibitory immunoglobulin G class 2 response against cFVII only in the dog receiving the highest AAV dose. In conclusion, in the only large-animal model representing the majority of FVII mutation types, our data are first to demonstrate the feasibility, safety, and long-term duration of AAV-mediated correction of FVII deficiency.

A sizable proportion of hemophilia inhibitor patients fails immune tolerance induction and requires bypass agents for long-term bleed management. Recombinant human-activated coagulation Factor VII (rhFVIIa) is an on-demand bypass hemostatic agent for bleeds in hemophilia inhibitor patients. Prophylactic use of rhFVIIa may enable sustained hemostatic management of inhibitor patients, but the critical relationship of rhFVIIa circulating levels and clinical outcome in that setting remains unclear. To address this in vivo, we used the rat hemophilia A (HA) model that exhibits spontaneous bleeds and allows longitudinal studies with sufficient statistical power. We simulated activated Factor VII (FVIIa) prophylaxis by adeno-associated virus (AAV) gene transfer of a rat FVIIa transgene. Compared with naive HA animals, rat FVIIa continuous expression affected the overall observed bleeds, which were resolved with on-demand administration of recombinant rat FVIIa. Specifically, although 91% of naive animals exhibited bleeds, this was reduced to 83% and 33% in animals expressing less than 708 ng/mL (25 nM). Rat FVIIa expression of at least 708 ng/mL was also sufficient to normalize the blood loss after a tail vein injury. Continuous, AAV-mediated rat FVIIa transgene expression had no apparent adverse effects in the hemostatic system of HA rats. This work establishes for the first time a dose dependency and threshold of circulating FVIIa antigen levels for reduction or complete elimination of bleeds in a setting of FVIIa-based HA prophylaxis.

Methamphetamine (METH) substance abuse disorders have major impact on society, yet no medications have proven successful at preventing METH relapse or cravings. Anti-METH monoclonal antibodies can reduce METH brain concentrations; however, this therapy has limitations, including the need for repeated dosing throughout the course of addiction recovery. An adeno-associated viral (AAV)-delivered DNA sequence for a single-chain variable fragment could offer long-term, continuous expression of anti-METH antibody fragments. For these studies, we injected mice via tail vein with 1 x 10(12) vector genomes of two AAV8 scFv constructs and measured long-term expression of the antibody fragments. Mice expressed each scFv for at least 212 days, achieving micromolar scFv concentrations in serum. In separate experiments 21 days and 50 days after injecting mice with AAV-scFvs mice were challenged with METH in vivo. The circulating scFvs were capable of decreasing brain METH concentrations by up to 60% and sequestering METH in serum for 2 to 3 hrs. These results suggest that AAV-delivered scFv could be a promising therapy to treat methamphetamine abuse.

Summary Background The ability of the spliceosomal small nuclear RNA U1 (U1snRNA) to rescue pre-mRNA splicing impaired by mutations makes it an attractive therapeutic molecule. Coagulation factor deficiencies due to splicing mutations are relatively frequent and could therefore benefit from this strategy. However, the effects of U1snRNAs in vivo remain unknown. Objectives To assess the rescue of the F7 c.859+5G>A splicing mutation (FVII+5A), causing severe human factor VII (hFVII) deficiency, by the modified U1snRNA+5a (U1+5a) in a murine model. Methods Mice expressing the human F7 c.859+5G>A mutant were generated following liver-directed expression by plasmid or recombinant adeno-associated viral (AAV) vector administration. The rescue of the splice-site defective pre-mRNA by U1+5a was monitored in liver and plasma through hFVII-specific assays. Results Injection of plasmids encoding the U1+5a rescued plasma hFVII levels, which increased from undetectable to ~8.5% of those obtained with the wild-type hFVII plasmid control. To assess long-term effects, mice were injected with low and high doses of two AAV vectors encoding the FVII+5A splice site mutant as template to be corrected by U1+5a. This strategy resulted in hFVII plasma levels of 3.9 ± 0.8 or 23.3 ± 5.1 ng mL−1 in a dose-dependent manner, corresponding in patients to circulating FVII levels of ~1–4.5% of normal. Moreover, in both experimental models, we also detected correctly spliced hFVII transcripts and hFVII-positive cells in liver cells. Conclusions Here we provide the first in vivo proof-of-principle of the rescue of the expression of a splicing-defective F7 mutant by U1snRNAs, thus highlighting their therapeutic potential in coagulation disorders.

The endothelial protein C receptor (EPCR) has been demonstrated to bind activated FVII (FVIIa) through the Gla domain with equal affinity to Protein C (PC). Mouse studies suggest that EPCR is involved in the extravasation of infused human FVIIa, leading to an extended extravascular tissue persistence, longer than expected based on its circulating half-life. This provides a plausible explanation for the long-term benefits of hemophilic patients on human FVIIa prophylaxis. Collectively, these data suggest that EPCR sequesters administered FVIIa in tissues where it may have a hemostatic effect. However, the role of the endogenous FVII-EPCR interaction in normal conditions is largely unknown. For this, we have developed a mouse model to better understand this interaction in vivo. Endogenous mouse FVII and FVIIa (mFVII/FVIIa) do not bind mouse EPCR. However, our laboratory has demonstrated that L4F, L8M, and T9R substitutions in the Gla domain of mFVIIa enable its interaction with mouse EPCR while retaining full enzymatic activity in vitro. Based on that data, we utilized CRISPR/Cas9 technology to knock-in L4F, L8M, and T9R into the mFVII Gla domain in the mouse F7 locus (F7FMR), thereby developing mice with a chimeric endogenous FVII capable of binding EPCR. Founder animals were generated and capable of producing offspring, indicating that the gain-of-function in mFVII was compatible with life. Animals were subsequently backcrossed to wildtype C57BL/6 mice in order to remove potential off-target effects of the CRISPR/Cas9. Resultant heterozygous animals (F7FMR/WT) from the final cross were bred to generate F7FMR/FMR, F7FMR/WT, and F7WT/WTlittermates. We generated 59 male and 52 female animals and a binomial distribution test demonstrated that sex is equally distributed in the population. Moreover, the genotypes expected from the heterozygous crosses were inherited in a 1:2:1 ratio, further indicating that the gain-of-function in FVII is not lethal during development. As additional metrics of health, we measured weight longitudinally during weeks 1-10 of life and found no differences between the three genotypes for either gender. Complete blood counts (CBCs) revealed no differences between the F7FMR/FMR, F7FMR/WT, and F7WT/WTgenotypes, with the exception of a mild elevation in F7FMR/WTanimals compared to animals with wildtype FVII. Collectively, we found that the gain-of-function in EPCR binding by endogenous FVII is not detrimental to the overall health of the mice. Subsequently, we determined the mFVII levels in the F7FMR/FMR, F7FMR/WT, and F7WT/WTanimals using an in-house ELISA. We observed that plasmatic mFVII levels were dependent on the EPCR-binding capacity of the endogenous mFVII. Specifically, F7WT/WTmice, whose mFVII does not bind EPCR, had a plasmatic mFVII concentration of ~690 ng/ml. In contrast, F7FMR/FMRhomozygote mice had ~350 ng/ml of mouse FVII, approximately half the plasma levels of the F7WT/WT. Heterozygote animals F7FMR/WThad an intermediate plasmatic mFVII level (~550 ng/ml), suggesting that EPCR may regulate plasmatic FVII levels in vivo. Lastly, we determined the hemostatic response to injury in the F7FMR/FMR, F7FMR/WT, and F7WT/WTanimals. We did this in two ways, by measuring blood loss following tail clip assay and by determining time to vessel occlusion following ferric chloride injury of the carotid artery. We observed no differences between the three genotypes in response to either injury model. In conclusion, we have generated and characterized a novel mouse model in which endogenous FVII is capable of binding EPCR. Using this model, we demonstrated that EPCR can modulate plasmatic FVII levels in vivo but does not appear to affect hemostasis. Since this model mimics the FVII-EPCR interaction in humans, it can now be used to further investigate how this interaction participates in other normal or pathologic states that depend on FVII and/or EPCR. Disclosures Margaritis: Bayer Hemophilia Awards: Research Funding; Bristol-Myers Squibb: Other: Salary (spouse); CSL Behring: Other: Salary (spouse); NovoNordisk A/S: Research Funding.

Background: PRRG proteins were cloned more than a decade ago, but their function is still not known. Results: Several novel protein-protein interactions for PRRG are identified by array screening and pulldown analysis. Conclusion: PRRG-initiated signaling events most likely depend on proteins with WW domains. Significance: The protein-protein interactions identified here may help to elucidate the roles of PRRG proteins in different physiological settings., The genes encoding a family of proteins termed proline-rich γ-carboxyglutamic acid (PRRG) proteins were identified and characterized more than a decade ago, but their functions remain unknown. These novel membrane proteins have an extracellular γ-carboxyglutamic acid (Gla) protein domain and cytosolic WW binding motifs. We screened WW domain arrays for cytosolic binding partners for PRRG4 and identified novel protein-protein interactions for the protein. We also uncovered a new WW binding motif in PRRG4 that is essential for these newly found protein-protein interactions. Several of the PRRG-interacting proteins we identified are essential for a variety of physiologic processes. Our findings indicate possible novel and previously unidentified functions for PRRG proteins.

Essentials The lack of factor (F) VIIa-endothelial protein C receptor (EPCR) binding in mice is unresolved. A single substitution of Leu4 to Phe in mouse FVIIa (mFVIIa) enables its interaction with EPCR. mFVIIa with a Phe4 shows EPCR binding-dependent enhanced hemostatic function in vivo vs. mFVIIa. Defining the FVIIa-EPCR interaction in mice allows for further investigating its biology in vivo.Background Human activated factor VII (hFVIIa), which is used in hemophilia treatment, binds to the endothelial protein C (PC) receptor (EPCR) with unclear hemostatic consequences. Interestingly, mice lack the activated FVII (FVIIa)-EPCR interaction. Therefore, to investigate the hemostatic consequences of this interaction in hemophilia, we previously engineered a mouse FVIIa (mFVIIa) molecule that bound mouse EPCR (mEPCR) by using three substitutions from mouse PC (mPC), i.e. Leu4→Phe, Leu8→Met, and Trp9→Arg. The resulting molecule, mFVIIa-FMR, modeled the EPCR-binding properties of hFVIIa and showed enhanced hemostatic capacity in hemophilic mice versus mFVIIa. These data implied a role of EPCR in the action of hFVIIa in hemophilia treatment. However, the substitutions in mFVIIa-FMR only broadly defined the sequence determinants for its mEPCR interaction and enhanced function in vivo. Objectives To determine the individual contributions of mPC Phe4, Met8 and Arg9 to the in vitro/in vivo properties of mFVIIa-FMR. Methods The mEPCR-binding properties of single amino acid variants of mFVIIa or mPC at position 4, 8 or 9 were investigated. Results and conclusions Phe4 in mFVIIa or mPC was solely critical for interaction with mEPCR. In hemophilic mice, administration of mFVIIa harboring a Phe4 resulted in a 1.9-2.5-fold increased hemostatic capacity versus mFVIIa that was EPCR binding-dependent. This recapitulated previous observations made with triple-mutant mFVIIa-FMR. As Leu8 is crucial for hFVIIa-EPCR binding, we describe the sequence divergence of this interaction in mice, now allowing its further characterization in vivo. We also illustrate that modulation of the EPCR-FVIIa interaction may lead to improved FVIIa therapeutics.

Activated FVII (FVIIa) has been shown to interact with EPCR resulting in long-term extravascular tissue presence in mice, well beyond its short circulating half-life. In addition, data in hemophilia patients on short term FVIIa prophylaxis indicate that clinical improvements can persist even in the post-prophylaxis period. Taken together, these data suggest that EPCR may sequester administered FVIIa in the extravascular space that retains activity, potentially explaining the hemostatic improvements that persist long after its infusion has ceased. As such, the EPCR-FVIIa interaction may have mechanistic and translational ramifications in the treatment of bleeds in hemophilia which needs to be further investigated. Unfortunately, hemophilic mice do not model the human disorder in terms of bleeding diathesis. In contrast, the novel rat model of hemophilia A (Nielsen LN et al, 2014) exhibits spontaneous bleeds that, if untreated, can be fatal. Therefore, this model can be utilized to better understand the FVIIa-EPCR interaction in the on-demand and prophylactic action of FVIIa administration in hemophilia. As an initial step towards that goal, here we characterize for the first time the rat FVIIa-EPCR interaction in vitro. We generated rat FVIIa by introduction of a PACE/Furin intracellular cleavage site (RKRRKR) between the light and heavy chains of rat FVII, an approach we have previously shown to result in secretion of two-chain activated FVII protease of various species (human, mouse and canine) in vitro. Recombinant rat FVIIa was purified in a two-chain form using conditioned medium from a HEK-293 stable cell line. The affinity of rat FVIIa to rat EPCR was assessed by incubation of the protease on engineered CHO-K1 cells that stably expressed full length rat EPCR. We found that rat FVIIa exhibited minimal binding to rat EPCR; in contrast human FVIIa bound rat EPCR with a Kd of ~400 nM. The lack of EPCR binding of rat FVIIa, similar to what we previously observed with mouse FVIIa (Pavani G et al., 2014 and 2016), complicates studies aimed at deciphering the hemostatic effects of the FVIIa-EPCR interaction in hemophilia using the rat. Therefore, we decided to engineer a rat FVIIa molecule with EPCR binding capacity so that it can be compared to rat FVIIa (non-EPCR binder) in subsequent in vivo experiments. For this, a rat FVIIa chimeric protease was generated using the first 11 amino acids of the Gla domain of rat protein C (rat FVIIa-FVRAG, with substitutions L4F, L8V, W9R, S10A, S11G). Using CHO-K1 expressing rat EPCR, we found that purified recombinant rat FVIIa-FVRAG bound rat EPCR with a Kd of ~500 nM, comparable to human FVIIa. Considering that the Gla domain, where the modifications are located, is essential for protease function, we also determined the procoagulant activity of rat FVIIa-FVRAG vs. FVIIa. We performed a thrombin generation assay in which each protease was added at increasing concentration (0-500 nM) and we measured the lag time, peak thrombin, endogenous thrombin potential. We found that rat FVIIa, rat FVIIa-FVRAG and human FVIIa (control) behaved similarly in all measured parameters for each concentration tested. In conclusion, we have defined the lack of EPCR binding of rat FVIIa and characterized a recombinant rat FVIIa molecule that can bind rat EPCR in vitro as a gain of function, but is otherwise similar to rat FVIIa. This molecule can now be used to probe the FVIIa-EPCR interaction in the novel and relevant hemophilia A rat model. Disclosures Margaritis: Bayer Hemophilia Awards: Research Funding; Bristol Myers Squibb: Other: Salary (spouse); Novo Nordisk A/S: Research Funding.

Dogs with haemophilia A or haemophilia B exhibit spontaneous bleeding comparable with the spontaneous bleeding phenotype that occurs in humans with severe haemophilia. The phenotypic and genotypic characteristics of haemophilic dogs have been well-described, and such dogs are suitable for testing prophylactic protein replacement therapy and gene transfer strategies. In dogs with haemophilia, long-term effects on spontaneous bleeding frequency (measured over years) can be used as an efficacy endpoint in such studies. Although complete correction of coagulopathy has not been achieved, published data show that prophylactic factor replacement therapy and gene transfer can markedly reduce the frequency of spontaneous bleeding in haemophilic dogs. Further studies are currently ongoing.

Traditional treatment for haemophilia consists of bolus infusion of the missing coagulation factor, either prophylactically or on demand, but is complicated by the development of inhibitory antibodies to the infused factor. In those cases, as well as in patients with platelet defects or factor VII (FVII) deficiency, recombinant human activated FVII has been successfully used, but carries the disadvantage of a short plasma half-life. As an alternative, emerging methodology based on gene transfer may be utilized to provide effective haemostasis in patients with coagulation defects. The goal of this article is to introduce the novel concept of continuous expression of activated FVII from a donated gene for the treatment of haemophilia, and to review the safety and efficacy data that have been produced so far by this approach in small and large animal models.

Continuous expression of activated factor VII (FVIIa) via gene transfer is a potential therapeutic approach for hemophilia patients with or without inhibitory antibodies to human factor VIII (FVIII) or IX (FIX). Here, we investigate whether gene transfer of an engineered canine FVIIa (cFVIIa) transgene can affect hemostasis in a canine model of hemophilia, a good predictor of efficacy of hemophilia treatments. Purified recombinant cFVIIa exhibited 12-fold higher tissue factor–dependent activity than purified recombinant zymogen cFVII. Subsequently, we generated a serotype 8 recombinant adeno-associated viral vector expressing cFVIIa from a liver-specific promoter. Vector delivery via the portal vein in hemophilia A and B dogs was well tolerated, and long-term expression of cFVIIa resulted in a shortening of the prothrombin time, partial correction of the whole blood clotting time and thromboelastography parameters, and a complete absence of spontaneous bleeding episodes. No evidence of hepatotoxicity, thrombotic complications, or inhibitory immune response was found. These data provide the first evidence for in vivo efficacy and safety of continuously expressed FVIIa as a FVIII/FIX-bypassing agent in a large animal model of hemophilia, avoiding the risk of inhibitor formation associated with bolus FVIII or FIX infusion.

Intravenous infusion of recombinant human activated Factor VII (FVIIa) has been used for over a decade in the successful management of bleeding episodes in patients with inhibitory antibodies to Factor VIII or Factor IX. Previously, we showed that expression of murine FVIIa (mFVIIa) from an adeno-associated viral (AAV) vector corrected abnormal hemostatic parameters in hemophilia B mice. To pursue this as a therapeutic approach, we sought to define safe and effective levels of FVIIa for continuous expression. In mice transgenic for mFVIIa or injected with AAV-mFVIIa, we analyzed survival, expression levels, in vitro and in vivo coagulation tests, and histopathology for up to 16 months after birth/mFVIIa expression. We found that continuous expression of mFVIIa at levels at or below 1.5 microg/ml was safe, effective, and compatible with a normal lifespan. However, expression levels of 2 microg/ml or higher were associated with thrombosis and early mortality, with pathologic findings in the heart and lungs that were rescued in a low-factor X (low-FX) mouse background, suggesting a FX-mediated effect. The findings from these mouse models of continuous FVIIa expression have implications for the development of a safe gene transfer approach for hemophilia and are consistent with the possibility of thromboembolic risk of continuously elevated FVIIa levels.

Summary. Background: Canine factor VII (cFVII) deficiency, an autosomal recessive trait originally identified in research Beagles, is associated with a mild to moderate bleeding tendency. Objective: Our aim was to identify and characterize the mutation causing cFVII deficiency. Methods: In order to sequence the coding regions of the cFVII gene, we cloned the cFVII cDNA. Genomic DNA and plasma from FVII-deficient Beagles and obligate carriers were utilized. Results: In all FVII-deficient dogs, we identified a single causative G to A missense mutation in exon 5, encoding the second epidermal growth factor-like domain, resulting in substitution of glycine 96 by glutamic acid, with plasma FVII coagulant activity of ≤ 4% in affected Beagles. In vitro expression indicated that the majority (96%) of cFVII-G96E protein was retained intracellularly. In addition, analysis of purified recombinant wild-type and mutant cFVII proteins demonstrated reduced activity of the mutant (

Hemophilia is a bleeding disorder caused by mutations in the genes encoding coagulation Factor VIII (FVIII) or FIX. Current treatment is through intravenous infusion of the missing protein. The major complication of treatment is the development of neutralizing Ab’s to the clotting factor. Infusion of recombinant activated human Factor VII (rhFVIIa), driving procoagulant reactions independently of human FVIII (hFVIII) or hFIX, has been successful in such patients and could in theory provide hemostasis in all hemophilia patients. However, its high cost and short half-life have limited its use. Here, we report a novel treatment strategy with a recombinant adeno-associated virus vector delivering a modified FVII transgene that can be intracellularly processed and secreted as activated FVII (FVIIa). We show long-term expression, as well as phenotypic correction of hemophilia B mice following gene transfer of the murine FVIIa homolog, with no evidence of thrombotic complications at these doses. These data hold promise for a potential treatment for hemophilia and other bleeding disorders.

Recombinant activated human factor VII (rhFVIIa) is an established hemostatic agent in hemophilia, but its mechanism of action remains unclear. Although tissue factor (TF) is its natural receptor, rhFVIIa also interacts with the endothelial protein C receptor (EPCR) through its γ-carboxyglutamic acid (Gla) domain, with unknown hemostatic consequences in vivo. Here, we study whether EPCR facilitates rhFVIIa hemostasis in hemophilia using a mouse model system. Mouse activated FVII (mFVIIa) is functionally homologous to rhFVIIa, but binds poorly to mouse EPCR (mEPCR). We modified mFVIIa to gain mEPCR binding using 3 amino acid changes in its Gla domain (L4F/L8M/W9R). The resulting molecule mFVIIa-FMR specifically bound mEPCR in vitro and in vivo and was identical to mFVIIa with respect to TF affinity and procoagulant functions. In macrovascular injury models, hemophilic mice administered mFVIIa-FMR exhibited superior hemostatic activity compared with mFVIIa. This was abolished by blocking mEPCR and was absent in ex vivo whole blood coagulation assays, implicating a specific mFVIIa-FMR and endothelial mEPCR interaction. Because mFVIIa-FMR models the TF-dependent and EPCR binding properties of rhFVIIa, our data unmask a novel contribution of EPCR on the action of rhFVIIa administration in hemophilia, prompting the rational design of improved and safer rhFVIIa therapeutics.

In this issue of Blood , Feng et al shed incisive light on how recombinant activated factor VII (rFVIIa) corrects the bleeding defect in hemophilia.[1][1] ![Figure][2] The different proposed models to explain rFVIIa dosing in hemophilia. (A) In the TF-dependent model, high doses of rFVIIa (

The traditional treatment for hemophilia has been by protein replacement. This is complicated by the development of inhibitory antibodies to the infused factor (Factor VIII [FVIII] or Factor IX [FIX]). High-dose infusion of recombinant activated Factor VII (rFVIIa) has a long track record of success in such patients but its short-half life limits its use in prophylaxis. We have developed an alternative strategy by continuous expression of activated FVII from a transgene that is introduced into the host by means of gene transfer. For this, we modified the FVII cDNA to introduce a cleavage site between the light and heavy chain that would generate a FVII molecule secreted in the two-chain, activated form. Using viral-mediated delivery and expression from a liver-specific promoter (or as a transgenic approach) we demonstrated the long-term hemostatic efficacy of this approach in hemophilic mice. Subsequently, we used the canine version of our modified FVII and via gene transfer, showed multi-year phenotypic correction in hemophilic dogs, clearly evident by the absence of spontaneous bleeds that are characteristic in this animal model. No adverse events were observed throughout the study. Remarkably, clinical benefit was also observed in one treated dog despite the lack of hemostatic effect by in vitro assays. Overall, the results in this large animal model of hemophilia indicate the potential of gene-based continuous expression of activated FVII as a therapeutic strategy for hemophilia or other coagulation defects currently treated by rFVIIa.

Although recombinant factor VIIa has been shown to bypass the deficiency in factor VIII or factor IX, efforts to reduce factor consumption and subsequently treatment cost have been focused on continuous infusion regimens with variable success. Using an engineered factor VII that can be secreted as factor VIIa, viral-mediated delivery of this transgene in the mouse liver resulted in phenotypic correction of murine hemophilia B, at expression levels of approximately 1 microg/ml. This model of factor VIIa continuous infusion can be further used to address the potential risk of thrombosis, in a transgenic model approach. The relative contribution of the extrinsic and/or intrinsic pathways in such risk can be dissected by crossing over-expressing FVIIa mice to recently described models of low expression of tissue factor or factor X. Our current data support the potential of factor VIIa gene transfer as a therapeutic alternative to bolus dosing but effective monitoring and modulation of factor VIIa expression will most likely be required.

Qualitative or quantitative defects in the genes for coagulation factors VIII (FVIII) or IX (FIX) result in a life-threatening, bleeding phenotype (hemophilia A (HA) or B (HB), respectively). Although hemophilia treatment by clotting factor replacement is effective, a proportion of patients develop neutralizing antibodies (inhibitors) to the infused factor that complicate the disease management. For inhibitor patients, recombinant human activated coagulation Factor VII (rhFVIIa), when administered at therapeutic doses, has been shown to bypass the deficiency in FVIII or FIX and result in hemostasis. As an alternative to this protein infusion therapy, a gene-based approach for the treatment of hemophilia with inhibitors has been developed, using continuous expression of a transgene coding for FVIIa following viral-mediated delivery. This approach was validated in hemophilic mice and, notably, in dogs as a model that closely resembles the human disease. In particular, liver-directed FVIIa gene delivery in hemophilic dogs resulted in multi-year transgene expression that ameliorated the bleeding phenotype, without thrombotic complications. These data support the gene-based FVIIa expression as a novel bypass therapy for hemophilia with inhibitors.

Severe coagulant factor VII (FVII) deficiency in postpubertal dizygotic twin males results from two point mutations in the FVII gene, a promoter region T→C transition at -60 and a His-to-Arg substitution at amino acid 348; both mutations prevent persistence of plasma functional FVII. This report documents longitudinal laboratory measurements from infancy to adulthood of FVII coagulant activity (FVII:C) in the twin FVII-deficient patients; it also details specific biochemical analyses of the -60 T→C mutation. The results revealed FVII:C levels of less than 1% in infancy that remain severely decreased through puberty and into adulthood. In-vitro analyses utilizing hepatocyte nuclear factor 4α (HNF4α) co-transfection and a chromatin immunoprecipitation assay indicate that the -60 T→C mutation severely diminishes functional interaction between the FVII promoter and transcription factor HNF4α. The importance of interaction between the FVII gene and HNF4α in normal FVII expression provides an in-vivo illustration of the regulated expression of an autosomal gene encoding a coagulation protein. The constancy of FVII:C and peripubertal patient symptomatology reported here illustrates androgen-independent expression in contrast to expression with an analogous mutation in the promoter region of the gene encoding coagulation FIX.

A validated method for assessing hemostasis in vivo is critical for testing the hemostatic efficacy of therapeutic agents designed for patients with bleeding disorders such as von Willebrand disease (VWD) and hemophilia A. We hypothesize that rate of bleeding and time to hemostasis can be monitored in vivo by acoustic radiation force impulse (ARFI) ultrasound. We performed ARFI imaging following 12-gauge needle puncture of hind limb muscle encompassing an ∼2 mm vein in six normal, eight naive hemophilia A before and after infusing canine factor VIII, three hemophilia A expressing canine factor VIIa following gene transfer, and two naive VWD dogs. Serial data sets were processed with custom software to (1) estimate the rate of hemorrhage and (2) estimate the time of hemostasis onset. The rate of hemorrhage during the first 30 min following puncture was markedly increased in the VWD dogs relative to normal but was not significantly different between normal, naive hemophilia A or hemophilia A expressing cFVIIa. ARFI-derived times to hemostasis were significantly longer in naive hemophilia A dogs than in normal dogs and were shortened by canine coagulation factors VIII and VIIa. These data support our hypothesis that rate of hemorrhage and time to hemostasis in vivo in response to a standardized hemostatic challenge can be detected by ARFI ultrasound in canine models of VWD and hemophilia. These data also suggest that the ARFI-monitored hemostatic challenge is relevant for in vivo testing of the hemostatic efficacy of therapeutic clotting factor replacement products used to treat inherited bleeding disorders.

Catalytic domain variants of activated factor VII (FVIIa) with enhanced hemostatic properties are highly attractive for the treatment of bleeding disorders via gene-based therapy. To explore this in a hemophilic mouse model, we characterized 2 variants of murine activated FVII (mFVIIa-VEAY and mFVIIa-DVQ) with modified catalytic domains, based on recombinant human FVIIa (rhFVIIa) variants. Using purified recombinant proteins, we showed that murine FVIIa (mFVIIa) and variants had comparable binding to human and murine tissue factor (TF) and exhibited similar extrinsic coagulant activity. In vitro in the absence of TF, the variants showed a 6- to 17-fold enhanced proteolytic and coagulant activity relative to mFVIIa, but increased inactivation by antithrombin. Gene delivery of mFVIIa-VEAY resulted in long-term, effective hemostasis at 5-fold lower expression levels relative to mFVIIa in hemophilia A mice or in hemophilia B mice with inhibitors to factor IX. However, expression of mFVIIa-VEAY at 14-fold higher than therapeutic levels resulted in a progressive mortality to 70% within 6 weeks after gene delivery. These results are the first demonstration of the hemostatic efficacy of continuous expression, in the presence or absence of inhibitors, of a high-activity gene-based FVIIa variant in an animal model of hemophilia.

Dogs with hemophilia A and hemophilia B recapitulate the severe bleeding phenotype that occurs in humans with these disorders. We have investigated the use of Acoustic Radiation Force Impulse (ARFI) imaging as a method for assessing hemostasis in vivo in dogs with hemophilia A. ARFI imaging was performed in 6 normal, 7 hemophilia A, and 4 hemophilic dogs expressing canine factor FVIIa following gene transfer. Bleeding was induced by needle puncture in muscle and a 1–2 mm vein, and subcutaneous bleeding was monitored with ARFI for 100 minutes. Results showed delayed time to hemostasis in the hemophilia A dogs, consistent with the bleeding phenotype, but lower times to hemostasis in the FVIIa expressing dogs. These data suggest that ARFI imaging could be a relevant new hemostatic challenge for characterizing hemophilic phenotype.

Summary. Background: Activated factor X (FXa) is a vitamin K-dependent serine protease that plays a pivotal role in blood coagulation by converting prothrombin to thrombin. There are no reports of humans with complete deficiency of FX, and knockout of murine F10 is embryonic or perinatal lethal. Objective: We sought to generate a viable mouse model of FX deficiency. Methods: We used a socket-targeting construct to generate F10-knockout mice by eliminating F10 exon 8 (knockout allele termed F10tm1Ccmt, abbreviated as ‘−’; wild-type ‘+’), and a plug-targeting construct to generate mice expressing a FX variant with normal antigen levels but low levels of FX activity [4–9% normal in humans carrying the defect, Pro343Ser, termed FX Friuli (mutant allele termed F10tm2Ccmt, abbreviated as F)]. Results: F10 knockout mice exhibited embryonic or perinatal lethality. In contrast, homozygous Friuli mice [F10 (F/F)] had FX activity levels of ∼5.5% (sufficient to rescue both embryonic and perinatal lethality), but developed age-dependent iron deposition and cardiac fibrosis. Interestingly, F10 (−/F) mice with FX activity levels of 1–3% also showed complete rescue of lethality. Further study of this model provides evidence supporting a role of maternal FX transfer in the embryonic survival. Conclusions: We demonstrate that, while complete absence of FX is incompatible with murine survival, minimal FX activity as low as 1–3% is sufficient to rescue the lethal phenotype. This viable low-FX mouse model will facilitate the development of FX-directed therapies as well as investigation of the FX role in embryonic development.

Aminoglycosides can bypass nonsense mutations and are the prototypic agents for translational bypass therapy (TBT). Initial results demonstrate the need for more potent drugs and an in vivo model system for quantitative assessment of TBT. Herein, we present an in vivo system for evaluating the efficacy of premature stop codon management therapies: in vivo quantitative stop codon management repli-sampling TBT efficacy assay (IQSCMaRTEA). Application of IQSCMaRTEA reveals that geneticin is much more efficacious in vivo than gentamicin. Treatment with geneticin elicits a multiday response, and residual F9 antigen can be detected after 3 weeks. These data demonstrate the utility of IQSCMaRTEA for evaluating drugs that bypass nonsense mutations. In addition, IQSCMaRTEA may be helpful for testing inhibitors of nonsense-mediated decay, as stop codon management therapy will sometimes require inhibition of nonsense-mediated decay and translational bypass of the nonsense mutation. Furthermore, geneticin, its metabolites, or better tolerated analogues should be evaluated as a general treatment with multiday response for severe genetic disease caused by nonsense mutation.

The human protein C (PC) interaction with the endothelial PC receptor (EPCR) is mediated through the PC Gla domain, via key amino acids Phe4 and Leu8. Specifically, substitution of Leu8 with Val from human prothrombin abolishes the PC-EPCR interaction. The Gla domain of human Factor VII (FVII) shares these positions with PC and, consequently, the EPCR binding capacity. In the mouse, a commonly used in vivo model, the sequence determinants of the Gla domain of mouse PC (mPC) interaction with mouse EPCR (mEPCR) are not known. Remarkably, mouse FVII (mFVII) and its activated form (mFVIIa) have poor affinity for mouse EPCR. We previously described a variant of mFVIIa (mFVIIa-FMR) that contained the Leu4->Phe, Leu8->Met and Trp9->Arg from the mPC Gla domain. We found that this molecule was functionally similar to mFVIIa and could bind mEPCR. Using mFVIIa-FMR as surrogate to study the mPC-mEPCR interaction, we highlighted the importance of the Phe4/Met8/Arg9 in the mPC-mEPCR interaction. We also found that mFVIIa-FMR had enhanced hemostatic properties when infused at 3 mg/kg after FeCl3 carotid artery injury in hemophilic mice (vessel occlusion was 2.5 times faster than mFVIIa). In order to further refine whether the mEPCR binding capacity of mPC is coordinated by any/all of Phe4/Met8/Arg9 positions, we previously generated single variants of mPC at these positions using the corresponding amino acids of mFVIIa (that has poor interaction with mEPCR). We found that Phe4 is the sole determinant of specificity of the mPC-mEPCR interaction. Moreover, when Phe4 was placed in mFVIIa, we found that mFVIIa-Phe4 had activity similar to mFVIIa and bound mEPCR on cells (or in solution to soluble mEPCR) with a Kd of ~350nM. This was of similar magnitude to the mEPCR affinity of a mFVIIa variant with the entire mPC Gla domain (~200 nM), indirectly suggesting that Phe4 determines both the specificity and affinity of mPC to mEPCR. Since mFVIIa-FMR showed improved hemostatic properties in vivo as a result of mEPCR binding, enhancing the EPCR-FVIIa binding may generate improved human FVIIa molecules for the treatment of bleeding. Here we wanted to provide proof-of-concept using limited Gla domain modifications. For this, we utilized mFVIIa-Phe4, a minimally modified mFVIIa molecule, described above. Specifically, hemophilia B animals were subjected to a 7.5% FeCl3 injury of their carotid artery for 2 minutes; after 10 minutes mice were infused with 3 mg/kg of mFVIIa or mFVIIa-Phe4. Time to vessel occlusion was determined by monitoring blood flow. Hemostatically normal mice occluded in 13.3 ± 3.0 min. We found that infusion of mFVIIa resulted in vessel occlusion at 8.9 ± 1.7 min. However, mice that received mFVIIa-Phe4 reached vessel occlusion within 4.5 ± 2.4 min, ~2.5 times faster than mFVIIa-infused mice (P Disclosures Ivanciu: Bayer Hemophilia Awards Program: Research Funding. Margaritis:Novo Nordisk A/S: Research Funding; Bayer Hemophilia Awards Program: Research Funding.

The development of recombinant activated factor VII (rFVIIa; NovoSeven ® , Novo Nordisk, Bagsvaerd, Denmark) has provided an effective, alternative treatment strategy for hemophilia patients with inhibitors. However, its short half-life necessitates frequent infusions and results in high treatment costs. One potential solution to this problem may lie in the use of FVIIa gene transfer, which would achieve long-lasting therapeutic levels of expression from a single injection. Studies in animal models have shown that a recombinant adeno-associated viral vector can be used to insert both murine and human FVIIa into murine liver. Following FVIIa gene transfer, mice with hemophilia B demonstrated a long-term, dose-dependent increase in circulating levels of FVIIa, reduced prothrombin time, and correction of activated partial thromboplastin time into the normal range. In addition, blood loss following a modified tail-clip assay was significantly reduced. Ongoing studies in mice engineered to express a wide range of FVIIa levels aim to analyze organ histology and evaluate long-term survival, reproductive fitness, and real-time in vivo clot formation in the microvasculature. These studies are expanding our knowledge of the effects of continuously expressed rFVIIa, and it is hoped that they may eventually provide a new avenue for treatment of hemophilia.

Moderate to severe bleeding occurs in Bernard-Soulier syndrome (BSS) patients due to a rare inherited dysfunction of their platelet GPIb|[alpha]|/|[beta]|-IX-V receptor, a four-gene encoded protein complex. Defective receptor disrupts the interaction between platelets and vascular wall and form abnormal macrothrombocytopenia, in addition to causing impaired thrombin generation and defective platelet plug at the tissue injury site. While platelet transfusion is the conventional therapy, treatment with a bolus injection of recombinant human factor VIIa (rhVIIa, NovoSeven|[reg]|) offers an alternative strategy especially when alloimmunization develops against administered platelets. At pharmacological plasma levels, rhVIIa led to cessation of mild and severe bleeding episodes in a number of BSS patients (Ozelo et al., Ann Hematol, 84(12), 2005 and Almeida et al, Br J Haematol, 121(3) 2003). However, the short half-life of the protein and its high expense remain as two major disadvantages of a rhVIIa regimen. We here report a gene therapy-based approach resulting in continuous expression of activated murine factor VII (mVIIa) in the plasma of a mouse model for BSS. Male BSS mice were tail-injected with three doses of AAV8-mVIIa vector ranging from 0.3 to 4.8e12 vg/mouse. The liver specific expression of mVIIa resulted in a dose- dependent increase in plasma mVIIa levels and shortening of prothrombin time (PT) with a plateau at four weeks after injection. At mVIIa levels >500 ng/ml (n=5), there was a significant increase in the rate of initial and progressing clot formation in whole blood measured by a thromboelastogram (ROTEM|[reg]|) compared to untreated age- and gender-matched control mice (n=5); CT (266|[plusmn]|44 vs 387|[plusmn]|59, P

Adeno-associated viral (AAV) vectors are used for in vivo gene transfer in a number of preclinical models of genetic diseases (including large-animal models) and are currently being tested in clinical trials for treatment of hemophilia B and cystic fibrosis. Protocols for production of AAV vectors in a helper virus-free system are available and are based on transient transfection of HEK-293 cells with multiple plasmids. Scale-up of vector production has been labor intensive and inefficient because of a lack of larger culture vessels suitable for growth of adherent cells, large-scale transfection, and vector production. Here we report efficient production of AAV vector in roller bottles, which represents a 10-fold scale-up from the conventional flask or plate method. Optimized production yielded greater than 1013 vector genomes per bottle and was as cost effective as published protocols using plates. Successful vector production by this method was dependent on optimization of transfection by calcium phosphate precipitation, of monitoring of cell growth (by measurement of glucose consumption), of cell culture conditions, and CO2/air exchange with the culture vessel.

Post-translational modifications of coagulation factors in the liver are essential for function. The vitamin K dependent coagulation proteins (VKCPs) require vitamin K to undergo gamma carboxylation of the glutamic residues in their Gla domain by gamma-glutamyl carboxylase [GGCX]. The vitamin K is then recycled by the action of epoxide reductase [VKORc1] and/or quinone reductase [NQO1]. The hemostatic importance of the vitamin K “cycle” is evidenced by patients who may suffer bleeding complications when anticoagulated with warfarin, which targets the vitamin K cycle. Moreover, the ability of a variety of VKCPs to secrete a biologically active product depends on the removal of their propeptide by the action of the intracellular endoprotease furin [FURIN gene]. Previous in vitro work on recombinant coagulation Factor IX, which is used for hemophilia B treatment, has connected these two processing steps by showing that endogenous VKORc1 as well as FURIN can be limiting factors in high-yield expression systems. In vivo, skeletal muscle (in contrast to liver) has been utilized to express low levels of coagulation Factor IX in the first hemophilia B gene therapy clinical trial. However, our experiments in mice demonstrated that the specific activity of muscle-synthesized Factor IX via gene transfer decreased at the high levels of FIX expression by a limited muscle area (Schuettrumpf J. et al., Blood 2005). These results suggest that in vitro and in vivo expression of biologically-active VKCPs outside the liver may be limited by the host cell post-translational modification machinery. Here, we performed a systematic study to determine the expression profiles of the vitamin K cycle and furin endoprotease genes in human liver and muscle, compared to the mouse. We also established these profiles in two hemophilic dogs, given the extensive use of this animal model in gene-based hemophilia therapies. RNA from liver and skeletal muscle was used as a template for reverse transcription and the subsequent relative quantification of the GGCX, VKORc1, NQO1, and FURIN genes by qPCR in each tissue using a housekeeping reporter gene. For this, a variety of housekeeping genes were investigated in all three species to identify ones with similar transcript levels in both liver and muscle tissue. We identified the housekeeping genes HPRT1, beta actin, and 18s rRNA as equivalently expressed in the liver and skeletal muscle of human, mouse, and dog, respectively. The relative mRNA transcript quantification of the vitamin K cycle genes in humans showed that the transcript levels of GGCX were similar in liver and muscle. In contrast, both VKORc1 and NQO1 were under-expressed in muscle vs. liver (69.5 ± 4.9% and 67.8 ± 12.5%, respectively, P Disclosures No relevant conflicts of interest to declare.

The Endothelial Protein C Receptor (EPCR) is an important component of the Protein C anticoagulant pathway. The interaction of Protein C (PC) through its Gla domain with EPCR enhances PC activation, thus down-regulating thrombin production. EPCR can also bind human activated Factor VII (FVIIa) and modulate its activity and localization. The residues involved in receptor recognition in both PC and FVIIa are Phe4 and Leu8, located in the first portion of the Gla domain. The importance of Phe8 is indicated by the lack of EPCR binding of a PC variant that contains a Val8 from human prothrombin. Remarkably, the similarity between PC and FVIIa for EPCR binding is lacking in the mouse. Others and we have shown that mouse FVIIa (mFVIIa, which contains a Leu4 and a Leu8) interacts with mouse EPCR (mEPCR) poorly, thus failing to model the spectrum of known human FVIIa properties. In previous work, we generated mFVIIa chimeras that contain parts of the mouse PC (mPC) Gla domain and determined that three residues in the mPC Gla domain can confer mEPCR binding to mFVIIa. Specifically, molecule mFVIIa-FMR that contained the Leu4->Phe, Leu8->Met and Trp9->Arg from mPC was functionally similar to mFVIIa and could bind mEPCR as a true gain-of-function. However, little is known on the contribution of any/all of these positions in mPC binding to mEPCR. Here, we wanted to understand the sequence determinants that dictate this interaction. For this, we generated single amino acid mutants of mPC at position 4, 8 or 9 from the corresponding residues of mFVIIa. Using conditioned medium from transiently transfected cells, we tested the ability of each mPC mutant to bind to mEPCR expressed on the surface of CHO-K1 cells. A single substitution of Phe4 with Leu abolished mEPCR binding of mPC, in contrast to modifications at position 8 (Met to Leu) or 9 (Arg to Trp). The importance of Phe4 for the mPC-mEPCR interaction was confirmed in a reverse experiment modifying mFVIIa (that has poor mEPCR affinity) individually at position 4 (Leu to Phe), 8 (Leu to Met) or 9 (Trp to Arg) according to the mPC sequence. We found that Leu4->Phe was the sole modification that could confer mEPCR binding to mFVIIa. We have previously shown that the interaction of mFVIIa-FMR with mEPCR enhances its hemostatic function (vs. mFVIIa) after administration in hemophilic mice that have undergone injury (Pavani G et al, Blood 2014). To further explore the contribution of position 4 (Leu->Phe [L4F]) in these effects, recombinant mFVIIa-L4F was purified. Titration of mFVIIa-L4F on CHO-K1 cells expressing mEPCR showed a specific and dose-dependent receptor binding, in contrast to mFVIIa, confirming our previous data (see above). Moreover, mFVIIa-L4F showed no difference in clotting activity compared to mFVIIa in a prothrombin time-based assay. In order to compare mFVIIa-L4F to mFVIIa in its ability to generate mouse thrombin, we used a thrombin generation assay using hemophilia B plasma spiked with either procoagulant. We found that addition of either mFVIIa or mFVIIa-L4F generated similar amounts of thrombin at all concentrations tested (3.1 - 25 nM). Therefore, mFVIIa-L4F exhibited similar coagulant activity to mFVIIa but gained mEPCR binding capacity, a feature shared with mFVIIa-FMR. Further experiments are underway to determine whether the single substitution in mFVIIa-L4F is sufficient to recapitulate the enhanced hemostatic properties observed in vivo with mFVIIa-FMR. In conclusion, our findings identify a single amino acid residue (Phe4) in the Gla domain of mouse PC that plays a critical role in the binding to its natural receptor. This property can also be transplanted into mFVIIa, without affecting its coagulant activity. These observations reveal another difference between human and mouse systems and may have implications for EPCR-dependent functions or properties of other vitamin K-dependent proteins. Disclosures No relevant conflicts of interest to declare.

A persistent obstacle that has hampered gene transfer experiments is the short-term nature of transgene expression in vivo. In this article we present evidence for sustained expression from primary human keratinocytes, using the retroviral vector MFG. Primary keratinocytes were transduced in culture with the MFG retroviral vector containing the coding region from factor IX cDNA. Transduced keratinocytes, which secreted on average 830 ng of factor IX/10(6) cells/24 hr in tissue culture, were used to form a bilayered skin equivalent and grafted onto nude mice under a silicone transplantation chamber. Between 0.1 and 2.75 ng of human factor IX per milliliter was found in mouse plasma for more than 1 year, suggesting that keratinocyte stem cells were both transduced and grafted. The results show, for the first time, that long-term expression is obtainable in retrovirally transduced keratinocytes after transplantation.

Abstract 754 Background: The small nuclear RNA U1 (U1snRNA), the component of the U1snRNP with a key role in pre-mRNA splicing, is an attractive therapeutic molecule because it is able to rescue splicing impaired by mutations, often associated to human disorders. U1snRNA-mediated rescue of gene expression has been demonstrated in various cellular models of disease, including coagulation factor deficiencies. This approach maintains gene regulation in physiological tissues and overcomes limitations related to vector-mediated delivery of large genes. Although the U1snRNA-based strategy can be particularly beneficial for coagulation deficiencies in which even modest increase of functional protein levels would result in improvement of clinical phenotypes, no studies have been conducted in vivo. Here, to demonstrate the in vivo efficacy, we chose the F7 c.840+5G>A splicing mutation causing severe human factor VII (hFVII) deficiency and previously shown by us to be efficiently rescued in vitro by a modified U1snRNA variant, the U1+5a. Methods: Due to the absence of mouse models of FVII deficiency caused by splicing mutations, we created a novel in vivo model of human FVII deficiency by liver-restricted expression of the mutated hFVII splicing-competent cassette harboring the F7 c.840+5G>A mutation (FVII+5A). This allowed us to test whether co-expression of U1+5a, under the control of its endogenous promoter, can alleviate the splicing defect. The U1+5a-mediated rescue was assessed in C57BL/6 mice, either by transient (by hydrodynamic injection of plasmids) or prolonged (by adeno-associated viral [AAV] vectors) co-expression of the mutated hFVII minigene FVII+5A and of the U1+5a, driven by separate vectors. To avoid competition for AAV receptor binding, the FVII+5A and U1+5a expression cassettes were packaged into serotype 2 (AAV2-FVII+5A) and 8 (AAV8-U1+5a) vectors. The hFVII expression levels in mice were evaluated by human-specific RT-PCR and immunologic assays. Results: Co-delivery of plasmid pAAV-FVII+5A (1 or 2 μg/g of mouse body weight) with a molar excess (1.5X) of pAAV-U1+5a resulted in a dose-dependent increase of circulating hFVII levels (50.6±16.0 ng/ml or 178±126 ng/ml, respectively), with a peak of 367ng/ml, corresponding to 16.7% of those observed in mice injected with the pAAV-FVIIwt. In contrast, the pAAV-FVII+5A alone did not produce detectable hFVII protein levels. Immunohistochemical analysis of livers of mice receiving both pAAV-FVII+5A and pAAV-U1+5a clearly showed hFVII-positive cells. Moreover, we demonstrated restoration of hFVII splicing (26±10% of total transcripts) in hepatocytes, to indicate that the U1+5a efficiently re-directed usage of the mutated hFVII splicing site in vivo. To assess long-term effects of the U1+5a, mice were injected with 1.2 × 1012 vector genomes (vg)/mouse of AAV2-FVII+5A alone or with the AAV8-U1+5a (1.2×1011 or 6×1011 vg/mouse). In all co-transduced mice, we detected hFVII antigen levels in plasma, whereas delivery of AAV2-FVII+5A alone was ineffective. The U1+5a-mediated effect was dose-dependent, as measured by circulating hFVII levels of 3.9±0.8 ng/ml (1.2×1011 vg/mouse) or 23.3±5.1 ng/ml (6×1011 vg/mouse) at two weeks post-injection. These findings were corroborated by the appearance of correctly spliced hFVII transcripts (4±0.5% or 16±3% of the total transcripts, respectively) and of hFVII-positive staining cells in liver histological sections. However, the highest AAV8-U1+5a dose (6×1011 vg/mouse) resulted in mortality within two weeks, an observation that warrants further studies to define the underlying mechanism. It is worth noticing that the lowest AAV8-U1+5a dose (1.2×1011 vg/mouse) resulted in low but detectable and persistent circulating hFVII levels. Moreover, its correction effect was even more appreciable (6.9±1.7 ng/ml) in mice injected with an increased dose of template AAV2-FVII+5A (6×1012 vg/mouse), thus suggesting that in FVII deficient patients bearing splicing defects, expressing the target F7 pre-mRNA in all hepatocytes, the rescue would be likely more robust. Conclusions: We utilized a novel methodology to model human FVII deficiency caused by a splicing defect and to evaluate correction approaches in vivo. Our data provide the first in vivo proof-of-principle of the U1snRNA-mediated rescue of gene expression, and highlight its therapeutic potential in coagulation factor disorders. Disclosures: Pagani: RareSplice: co-founder of RareSplice, a start-up company of the University of Ferrara Other. Bernardi:RareSplice: Co-founder of RareSplice, a start-up company of the University of Ferrara Other. Pinotti:RareSplice: Co-founder of RareSplice, a start-up company of the University of Ferrara Other.

Abstract 3350 Cancer is frequently associated with activation of coagulation, and a procoagulant state facilitates tumor metastasis. Recent studies have suggested that the activated protein C (aPC) pathway plays a role in modulating tumor metastasis, and this protection likely requires both the anticoagulant and cytoprotective effects of aPC. Notably, our early work revealed that the inactive precursor, zymogen PC (zyPC), can even more effectively protect against metastasis. The aim of this study was therefore to explore mechanisms through which zyPC could prevent metastatic cancer progression in a murine cancer model. A liver gene transfer model using viral vectors was utilized to achieve a wide range of sustained expression of wildtype (WT) or mutant murine zyPCs. C57BL/6 experimental mice expressing stable levels of zyPCs and age and gender matched control mice receiving PBS were injected intravenously with 2.5×105 murine melanoma B16F10 cells, which metastasize to the lungs. After 3 weeks the number of pulmonary tumors was determined. Expression of WT zyPC in C57BL/6s decreased the rates of metastasis in a dose-dependent manner compared to PBS controls (p 50% of normal, n=6) did despite high rates of metastasis. These data clearly demonstrate the protective role of zyPC in tumor progression. We then tested modified zyPCs to identify the critical functions responsible for our observations in this tumor model. Two mutants with minimal anticoagulant function, R15Q and S195A, were generated. zyPC-R15Q is unable to dock to the thrombin-thrombomodulin complex active site and so cannot be converted to aPC. Compared to PBS controls (n=7), mice expressing zyPC-R15Q still showed a significant decrease in the number of tumor foci (p Next, we tested the importance of the main PC/aPC cellular receptors in our model. Binding to endothelial protein C receptor (EPCR) enhances activation of PC. We inhibited this binding by injecting anti-EPCR blocking antibody 1560 (J Thromb Haemost. 2005 3:1351) intraperitoneally one hour prior to the B16F10 cells. zyPC-expressing mice that received anti-EPCR antibody (n=22) still had a significant reduction in tumor rates compared to PBS controls (n=10; p Despite elevated circulating levels of PC, zyPC-expressing mice did not suffer from increased blood loss following tail clipping or show prolonged activated partial thromboplastin times (aPTTs) compared to hemostatically normal mice. In conclusion, zyPC protects against metastatic cancer progression in a dose-dependent manner. Our data show for the first time that this zyPC effect is independent of its anticoagulant function. Furthermore, protection is not mediated through EPCR or PAR1, suggesting an alternative pathway by which zyPC limits tumor progression. These findings suggest that WT zyPC and variants with little to no anticoagulant function are safe and efficacious in preventing metastatic cancer progression. Disclosures: Van Sluis: PCT patent pending: Protein C: A Zymogen for Anti-Cancer Treatment Patents & Royalties. High:PCT patent pending: Protein C: A Zymogen for Anti-Cancer Treatment Patents & Royalties. Spek:PCT patent pending: Protein C: A Zymogen for Anti-Cancer Treatment Patents & Royalties. Arruda:PCT patent pending: Protein C: A Zymogen for Anti-Cancer Treatment Patents & Royalties.

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.

Abstract 531 Generation of anticoagulant activated protein C (aPC) is enhanced by the presentation of protein C (PC) on the endothelial protein C receptor (EPCR) to the thrombin/thrombomodulin complex. Apart from its role in anticoagulation, EPCR also binds human coagulation Factor VIIa (FVIIa). However, the physiological consequences of this interaction either in vitro or in vivo are still unclear. In lieu of the widespread use of high-dose human FVIIa in hemophilic patients with anti Factor VIII or FIX antibodies as well as off-label applications, in vivo experimentation in appropriate mouse models is necessary to further define of the role of the FVIIa-EPCR interaction. Towards this, the aim of this study was to characterize the interaction of murine FVIIa with murine EPCR (mEPCR) in vitro. We used two novel assays that we have developed to study this interaction either in solution (by isothermal titration calorimetry [ITC]) or on the cell surface of mEPCR-expressing cells. The choice of ITC was based on its ability to provide a complete thermodynamic profile of protein-protein interaction in a single experiment: binding constant (K), stoichiometry (n), enthalpy (δH) and entropy (δS), parameters that cannot be determined with other methodologies in a single experiment. We first generated stable cells lines expressing recombinant murine soluble EPCR (msEPCR) or recombinant mFVIIa. The latter was generated as we have previously shown by insertion of a furin intracellular cleavage site between the light and heavy chains of murine FVII (mFVII). This resulted in a molecule secreted and purified in the activated form (mFVIIa) with ∼100% extrinsic activity vs. human FVIIa. Recombinant purified msEPCR had an apparent molecular weight of ∼46 kDa (vs. a predicted ∼26kDa size) which was consistent with extensive carbohydrate modifications. This was confirmed by expressing msEPCR in CHO cells modified to limit the size of the attached N-linked glycan chains: expressed msEPCR from such cells showed a reduction in its apparent molecular size (∼ 38kDa). Next, to validate ITC for the study the FVIIa-EPCR interaction, we used msEPCR and plasma-derived human PC (pd-hPC), a known msEPCR binder, that interacted with msEPCR with a Kd of ∼200nM, a stoichiometry of ∼1:1 and a ΔH and ΔS of −2733 ± 62 cal/mol and 21.3 cal/mol/deg, respectively. Initial ITC experiments with purified recombinant murine PC (mPC) also confirmed its binding to msEPCR. In contrast, we did not observe an interaction between recombinant mFVIIa and msEPCR. This finding was not due to protein degradation, as confirmed by Coomassie protein staining prior to and after ITC. Subsequently, to investigate the mFVIIa-msEPCR interaction in the context of a membrane-anchored murine EPCR, we generated a CHO-K1 cell line that expressed surface-exposed, full-length mEPCR, as verified by flow cytometry. These cells were incubated with pd-hPC or recombinant mFVIIa (50nM) in the presence of physiological Ca2+ (1.6mM) and Mg2+ (0.6mM) ion concentration. The bound protein was eluted with 10mM EDTA, electrophoresed and detected by western blotting. Despite the low femtomolar sensitivity of this assay (corresponding to 0.4% and 0.16% of total mFVIIa and pd-hPC used, respectively), we did not observe mFVIIa binding on mEPCR expressing CHO-K1 cells. Corroborating the ITC data, pd-hPC exhibited detectable binding to mEPCR expressing CHO-K1 cells. As a control, neither pd-hPC nor mFVIIa bound to naïve CHO-K1 cells (no mEPCR expression). In conclusion, we have established two novel assays to study the putative mFVIIa-mEPCR binding and clearly documented the lack of such interaction either in solution or on the cell-membrane. This is in good agreement with recently-published data obtained with surface plasmon resonance. In contrast to human FVIIa-EPCR, our results suggest that extrapolation of certain EPCR-dependent pathologic/physiologic processes using the mouse may be biased by species-specific effects. However, if the mEPCR binding capacity can be engineered to mFVIIa (as seen for human FVIIa-EPCR binding), such mFVIIa molecule(s) may facilitate the dissection of processes involving FVIIa and EPCR using the mouse as a human surrogate in vivo system. The assays developed here can easily assess such possibilities. Disclosures: Pavani: Bayer Healthcare: Research Funding. Margaritis:Bayer Healthcare: Research Funding.

Abstract 718 Recent evidence on the role of the protein C (PC) pathway in tumor progression of the experimental mouse melanoma model has revealed that inhibition of the cytoprotective effects of endogenous activated PC (aPC) enhances tumor cell extravasation, whereas exogenous administration of recombinant human APC has a protective effect. Moreover transgenic mice overexpressing endothelial PC receptor (EPCR) in tissue endothelium exhibit low rates of tumor metastasis. Here we report our findings in C57Bl/6 mice expressing murine forms of APC or zymogen PC by viral-mediated gene transfer. Vector-injected mice resulted in continuous expression of murine APC (mAPC) or PC (mPC), which reached plateau levels after week 2. On week 3, we administered B16F10 murine melanoma cells (3.5×10^5) intravascularly and analyzed the rates of lung metastasis 21 days later compared to age and gender matched saline-injected groups (control cohort=26 mice). We observed a dose-dependent protective effect of mAPC. Mice expressing mAPC at levels of 7.3 ± 1.5 ng/ml (n=8) or lower (determined by a functional ELISA-capture assay) did not differ from saline injected mice (that had baseline mAPC levels < 3 ng/ml). By increasing the vector dose, mAPC levels of 25.6 ± 4.8 ng/ml (n=16) to 118 ±6 ng/ml (n=10) reduced the numbers of lung metastasis compared to saline injected mice (p Disclosures: High: Genzyme, Inc: Consultancy, Patents & Royalties; Third Rock Ventures: Consultancy; PTC Therapeutics:; Amsterdam Molecular Therapeutics:; Sangamo Biosciences:; Novo-Nordisk: Consultancy; Shire, Inc.: Consultancy.

Abstract 2466 Poster Board II-443 Recombinant human activated Factor VII (rhFVIIa) is extensively used in the management of hemophilic inhibitor patients. As an alternative, using an adeno-associated viral (AAV) vector as a delivery vehicle for an engineered transgene for activated canine FVII (cFVIIa), our large animal studies in hemophilia A and B dogs demonstrated effective, long-term hemostasis and elimination of the bleeding episodes. However, the relatively high vector doses required for efficacy underscores the need for improvements in the delivery vector and/or the transgene itself. To address this, we decided to investigate whether changes in the catalytic domain of FVIIa can improve its coagulant activity, thus resulting in lowering of the effective vector dose. Using the murine version of our engineered FVIIa (mFVIIa) as a backbone, we generated a variant with four amino-acid substitutions in the catalytic domain of murine FVIIa (L305V/A314E/K337A/I374Y, mFVIIa-VEAY), based on an existing enhanced activity human FVIIa variant. As we have previously described (Amer. Soc. Hematol. Meeting 2006, #3276; Amer. Soc. Gene Cell Ther. Meeting 2008, #127), purified mFVIIa-VEAY exhibited 6-7 fold higher intrinsic coagulant activity than mFVIIa. The hemostatic properties relative to mFVIIa were determined following AAV-mediated gene delivery in hemophilia A (HA) mice. Administration of AAV-mFVIIa-VEAY at 40-100 fold lower vector dose (1.2 - 3E10 vector genomes [vg]/mouse) than AAV-mFVIIa (1.2E12 vg/mouse, High dose) resulted in ∼10-fold lower expression of the mFVIIa-VEAY (based on RNA transcript levels), but was sufficient to result in long-term (>12 weeks) normalization of the hemophilic activated partial thromboplastin time (aPTT), similar to that seen with high dose AAV-mFVIIa (P>0.05). In addition, it improved hemostasis similar to that seen with AAV-mFVIIa following in vivo hemostatic challenges (tail clip assay and ferric chloride carotid artery injury). As an extension of these studies, we have now investigated the ability of low-dose mFVIIa-VEAY gene delivery to improve hemostasis in the presence of inhibitory antibodies to human Factor IX (hFIX). Using an adjuvant, we developed a protocol for generating persistent (>3 months, ongoing) inhibitory antibodies to human Factor IX (hFIX) in hemophilia B (HB) mice. Using the low dose of 3E10 vg/mouse, we administered AAV-mFVIIa-VEAY in HB mice with high titer (7-30 Bethesda units [BU]) to hFIX. In agreement with our previous results, following gene transfer, we observed normalization of the hemophilic aPTT at the expression plateau (4 weeks [ongoing]; P>0.05 vs. wildtype mice; P0.05), HA mice treated with low-dose mFVIIa-VEAY (∼40ng/ml, P>0.05) or HA mice treated with AAV-mFVIIa (∼35ng/ml, P Disclosures: High: Novo Nordisk: Grant Review Panel.