Haemophilia Activities Research Articles

A bispecific antibody NXT007 exerts a hemostatic activity in hemophilia A monkeys enough to keep a nonhemophilic state

BackgroundEmicizumab, a factor (F) VIIIa-function mimetic bispecific antibody (BsAb) to FIXa and FX, has become an indispensable treatment option for people with hemophilia A (PwHA). However, a small proportion of PwHA still experience bleeds even under emicizumab prophylaxis, as observed in the long-term outcomes of clinical studies. A more potent BsAb may be desirable for such patients. ObjectivesTo identify a potent BsAb to FIXa and FX, NXT007, surpassing emicizumab by in vitro and in vivo evaluation. MethodsNew pairs of light chains for emicizumab’s heavy chains were screened from phage libraries, and subsequent antibody optimization was performed. For in vitro evaluation, thrombin generation assays were performed with hemophilia A plasma. In vivo hemostatic activity was evaluated in a nonhuman primate model of acquired hemophilia A. ResultsNXT007 exhibited an in vitro thrombin generation activity comparable to the international standard activity of FVIII (100 IU/dL), much higher than emicizumab, when triggered by tissue factor. NXT007 also demonstrated a potent in vivo hemostatic activity at approximately 30-fold lower plasma concentrations than emicizumab’s historical data. In terms of dose shift between NXT007 and emicizumab, the in vitro and in vivo results were concordant. Regarding pharmacokinetics, NXT007 showed lower in vivo clearance than those shown by typical monoclonal antibodies, suggesting that the Fc engineering to enhance FcRn binding worked well. ConclusionNXT007, a potent BsAb, was successfully created. Nonclinical results suggest that NXT007 would have a potential to keep a nonhemophilic range of coagulation potential in PwHA or to realize more convenient dosing regimens than emicizumab.

Clinical Implications of Discrepancy between One-Stage Clotting and Chromogenic Factor IX Activity in Hemophilia B.

Discrepancy in factor IX activity (FIX:C) between one-stage assay (OSA) and chromogenic substrate assay (CSA) in patients with hemophilia B (PwHB) introduces challenges for clinical management. To study the differences in FIX:C using OSA and CSA in moderate and mild hemophilia B (HB), their impact on classification of severity, and correlation with genotype. Single-center study including 21 genotyped and clinically characterized PwHB. FIX:C by OSA was measured using ActinFSL (Siemens) and CSA by Biophen (Hyphen). In addition, in vitro experiments with wild-type FIX were performed. Reproducibility of CSA was assessed between three European coagulation laboratories. FIX:C by CSA was consistently lower than by OSA, with 10/17 PwHB having a more severe hemophilia type by CSA. OSA displayed a more accurate description of the clinical bleeding severity, compared with CSA. A twofold difference between OSA:CSA FIX:C was present in 12/17 PwHB; all patients had genetic missense variants in the FIX serine protease domain. Discrepancy was also observed with diluted normal plasma, most significant for values below 0.10 IU/mL. Assessment of samples with low FIX:C showed excellent reproducibility of the CSA results between the laboratories. FIX:C was consistently higher by OSA compared with the CSA. Assessing FIX:C by CSA alone would have led to diagnosis of a more severe hemophilia type in a significant proportion of patients. Our study suggests using both OSA and CSA FIX:C together with genotyping to classify HB severity and provide essential information for clinical management.

Palmitoylethanolamide Attenuates Pain-like Behavior in Factor VIII Deficient Mice

Coagulation factor VIII (FVIII) deficiency gives rise to hemophilia A, which is characterized by spontaneous joint bleeding. Uncontrolled bleeding drives inflammation and release of noxious and neuropathic factors, accompanied by joint degeneration, arthropathy, joint/chronic pain and impairment in physical activity. Thus, an unmet need is to develop strategies to target pain and improve physical activity in hemophilia. Chronic joint pain observed in osteoarthritis (OA) shares features of inflammation and hemarthropathy with hemophilia joint pain. Palmitoylethanolamide (PEA) is being evaluated for joint pain in OA in an interventional clinical trial (NCT05406726), which builds upon a previous double-blind, placebo-controlled evaluation suggesting its safety, tolerability, and efficacy in alleviating OA joint pain. PEA is an endogenous lipid mediator that interacts with the endogenous cannabinoid system to alleviate chronic pain, inflammation and signs of neuropathy without the intoxicating effects of cannabinoids. We reported that PEA reduced chronic pain in a mouse model of sickle cell disease (Argueta et al., Blood 2021), which presents with release of cell free heme, consequent inflammation and tissue damage similar to hemophilia. Due to its ability to reduce clinical pain outcomes and its beneficial effects on inflammation, osteoclastogenesis, and neuropathy, PEA may provide substantial benefits as a novel treatment for pain in hemophilia. To test this hypothesis, we utilized FVIII-knockout (KO) mice, which recapitulate features of human hemophilia A including spontaneous bleeding and pain (Cooke et al., J Thromb Haemost 2019), to evaluate the impact of daily PEA treatment on nociceptive and nocifensive behaviors. We treated 12 wks old male control (B6129SF2/J) and FVIII-KO (B6;129S-F8tm1Kaz/J) mice (Jackson Laboratory, Bar Harbor, ME) intraperitoneally with 20 mg/kg/d PEA (Cayman Chemical, Ann Arbor, MI) for 7 days followed by 7 day withdrawal. Mice were weighed and examined for mechanical hyperalgesia by application of Von Frey monofilaments to the plantar surface of the hindpaws (10 repetitions per paw) and weight bearing on pressure-sensitive plates (Incapacitance Tester, Ugo Basile, Gemonio, Italy) at baseline (BL), then after PEA treatment at 1, 4, and 24 hours, days 3 and 7; and after discontinuation of treatment on days 10 and 14. We also measured spontaneous nocifensive behaviors (locomotion, rearing, and grooming) over a 2-minute period in homecage (Generic USB webcam; Streampix 7, Norpix, Montreal, Canada) at BL, days 7 and 14. PEA treatment reduced mechanical hyperalgesia (~60%, P<0.001) in FVIII-KO mice after 1 hour, which continued to improve over 24 hours and was maintained for 7 days of treatment. Following withdrawal, mechanical hyperalgesia was no longer alleviated. These data suggest increased sensory pain in FVIII-KO mice, and that PEA has an analgesic effect. PEA treatment improved stance instability in weight bearing test of FVIII-KO mice (~66%, P<0.05) after 4 hours, which continued to improve over treatment period and persisted after withdrawal, up to day 10. However, improved weight bearing regressed by day 14. Improved weight bearing even after withdrawal of the PEA for 3 days suggests a disease modifying effect perhaps via reduction in joint inflammation, modulation of arthropathy and/other mechanisms. Non-evoked, spontaneous nocifensive behaviors show associations (Pearson correlation, P<0.05) with weight bearing at BL, days 7 and 14 in FVIII-KO mice. Thus, nocifensive behaviors may be a less invasive, indirect measure of weight bearing and joint pain. Body mass was not significantly affected by treatment and subsequent withdrawal. We provide the first evidence that PEA may reduce mechanical hyperalgesia and improve weight bearing in a translational model of hemophilia A. Several clinical evaluations of PEA indicate its safety, even at relatively large doses, and its efficacy in reducing inflammation and pain, which include joint pain and damage observed in OA. Our observations and the availability of PEA as a dietary supplement underscore the need for clinical and preclinical evaluation of PEA for hemophilia pain.

Exploration of the minimum necessary FVIII level at different physical activity levels in pediatric patients with hemophilia A.

Physical activity can increase joint stability and reduce the risk of injury in hemophilia patients. There is limited clinical data on target trough FVIII levels during physical activity in hemophilia A patients. Hence, this study aimed to explore the target trough FVIII level required to avoid bleeding during different physical activities in hemophilia A patients. Patients with severe or moderate hemophilia A, who underwent pharmacokinetics (PK) tests at our center were enrolled in this study. Physical activities and clinical information such as bleeding were recorded. The FVIII level during physical activity was calculated by the WAPPS-Hemo. A total of 105 patients were enrolled in this study. A total of 373 physical activities were recorded, of which 57.6% (215/373) was low-risk activities and the remaining 42.4% (158/373) was medium-risk activities. Most common physical activities were bicycling (59.0%), swimming (43.8%), running (48.6%), and jumping rope (41.0%). The FVIII trough level of low-risk physical activity was 3.8 IU/dl (AUC = 0.781, p = 0.002) and moderate-risk physical activity was 7.7 IU/dl (AUC = 0.809, p < 0.001). FVIII trough levels [low-risk activities: 6.1 (3.1, 13.2) IU/dl vs. 7.7 (2.3, 10.5) IU/dl, moderate-risk activities: 9.6 (5.8, 16.9) IU/dl vs. 10.2 (5.5, 11.0) IU/dl] were not statistically different between the mild arthropathy group and the moderate-severe arthropathy group. Multiple bleeding risk tended to increase with physical activities classified as moderate-risk (OR [95% CI]: 3.815 [1.766-8.238], p = 0.001). The minimum necessary FVIII level increased with higher risk physical activity, irrespective of arthropathy.

Correlation between Slope 2 in Clot Waveform Analysis of Activated Partial Thromboplastin Time with Factor VIII Activity in Hemophilia A

Hemophilia A is an inherited factor VIII deficiency disease, related to X chromosome. Diagnosis of Hemophilia A is made based on Factor VIII assay. Nowadays, Hemophilia A therapy is by giving factor VIII concentrate, so that monitoring of this therapy must be done by examine Factor VIII activity, but examination of Factor VIII activity is currently still limited in facilities and quite expensive. One of activated partial thromboplastin time (aPTT) optical methods can provide information about every stage of coagulation through clot waveform analysis. Factor VIII activity can describe in slope 2 of clot waveform analysis, which deficiency of factor VIII will cause slope 2 slighter than normal, because the clot form is not optimal and the light transmission recorded at clot waves do not decrease maximally. The aim of this study was to analyze the correlation between slope 2 on the clot waveform analysis of the optical method on aPTT test with Factor VIII activity in hemophilia A subjects. This was a correlative observational study cross sectional study, conducted at Hasan Sadikin General Hospital Bandung in August 2018-September 2019. The subjects were member of Hemophilia A sufferers of West Java Hemophilia Society. The research subjects were assesed for Factor VIII activity and optical method of aPTT. Slope 2 calculated from the clot waveform analysis that formed in aPTT examination. This study involved 43 subjects, with a median age of 6 years, an age range of 1-45 years, and 51.2% of patients aged 6-17 years. The results of Factor VIII activity in this study had a median 0% with a range 0-25.9%, and the value of slope 2 had a median 1.0%T/sec with a range 0.5-3.5%T/sec. The correlation test between slope 2 and Factor VIII activity with 95% confidence interval using Spearman's correlation test showed very strong positive correlation which statistically significant (r = 0.854 and p &lt;0.001). Conclusion: there was a statistically significant very strong positive correlation between slope 2 on the clot waveform analysis of aPTT optical method test with the activity of Factor VIII in Hemophilia A.

Development of an Optimized rAAV2/6 Human Factor 8 cDNA Vector Cassette for Hemophilia a Gene Therapy

Hemophilia A, which is caused by a mutation in the Factor 8 (F8) gene resulting in a deficiency or lack of the Factor VIII (FVIII) protein, is the most common inherited bleeding disorder in humans with an estimated worldwide incidence of half a million people. The disorder is X-linked and occurs in approximately 1 in 5,000 males; however there is also a growing appreciation of the impact on carrier females having a single mutant allele, with at least 10% of hemophilia A female carriers having less than normal clotting activity. Even modest increases in Factor V III activity (>1% of normal) can have a positive impact on patient lives, thus making the disease an ideal candidate for liver-directed gene therapy. Recombinant AAV (rAAV) has been used extensively for nearly 20 years as a gene therapy vector in preclinical and clinical studies where rAAV delivery to non-dividing tissues such as liver, brain and muscle affords stable, long-term transgene expression. However, there has been a lag in the clinical translation of a rAAV gene therapy approach for Hemophilia A/human F8 (hF8) compared to Hemophilia B/human Factor 9 due to poor yields of rAAV encoding a F8 transgene at clinical scale, and a requirement for large doses of rAAV F8 vector to achieve therapeutically relevant levels of circulating human FVIII (hFVIII), with the attendant risk of inducing an AAV-directed immune response requiring transient immunosuppression. To address these issues we optimized a rAAV F8 cDNA vector cassette to improve both virus yields and liver-specific hFVIII expression. The rAAV F8 cDNA vector cassette optimization required multi-factorial modifications to the liver-specific promoter module, hF8 transgene, synthetic polyadenylation signal and vector backbone sequence. This iterative process resulted in improved vector yields at research scale and greater than five-fold improvement in vector yields at clinical scale using our proven manufacturing process. Administration of the optimized rAAV hF8 cDNA packaged in serotype AAV2/6 at a dose of ~7.2E+12 vg/kg to both wild type and Hemophilia A mice resulted in robust circulating hFVIII levels and activity (levels in wild type mice were 241.6% of normal, and activity in Hemophilia A mice were 330.9% of normal). An analysis of hF8 mRNA levels in different tissues following dosage with our optimized vector demonstrated that hF8 expression from the modified promoter module was restricted to the liver. Notably there was a striking impact on hemostasis in the Hemophilia A mice treated with the optimized rAAV hF8 cDNA, with a reduction in bleeding time from 38.3 minutes to 2.5 minutes in treated mice (n = 12, p-value < 0.0001), which is in line with bleeding times in wild type mice. Initial studies in non-human primates (NHPs) resulted in supraphysiological levels of circulating hFVIII with mean peak values of 400-600% of normal levels. A follow up dose-ranging study was performed in NHPs with a rAAV2/6 F8 cDNA vector manufactured using our GMP clinical manufacturing process. Administration of vector doses ranging from 6E+11 vg/kg to 6E+12 vg/kg resulted in therapeutic circulating levels of hFVIII that were 5% - 229% of normal levels. The peak circulating hFVIII levels achieved in this dose-ranging study using GMP clinical-scale vector exceeds the levels previously reported in NHPs by several fold on an AAV vector dose basis. The high potency of this enhanced rAAV F8 cDNA cassette could significantly reduce the dose required to achieve therapeutically relevant levels in human subjects and reduce the potential of developing immune responses to AAV capsid requiring immunosuppression. DisclosuresRiley:Sangamo BioSciences Inc: Employment. Boonsripisal:Sangamo BioSciences Inc: Employment. Goodwin:Sangamo BioSciences Inc: Employment. Garces:Sangamo BioSciences Inc: Employment. Ballaron:Sangamo BioSciences Inc: Employment. Tran:Sangamo BioSciences Inc: Employment. Kang:Sangamo BioSciences Inc: Employment. Zhang:Sangamo BioSciences Inc: Employment. Meyer:Sangamo BioSciences Inc: Employment. Greengard:Sangamo BioSciences Inc: Employment. Surosky:Sangamo BioSciences Inc: Employment. Ando:Sangamo BioSciences Inc: Employment. Lillicrap:bayer: Research Funding; biogen: Research Funding; CSL: Research Funding; Octapharma: Research Funding; Sangamo Biosciences Inc: Research Funding. Nichol:Sangamo BioSciences Inc: Employment. Holmes:Sangamo BioSciences Inc: Employment.

In vivo efficacy of human recombinant factor IX produced by the human hepatoma cell line HuH-7.

Post-translational modifications of the CHO-cell-derived-recombinant human factor IX (FIX) currently used for the treatment of hemophilia B (HB) are different from plasma derived FIX. Our previous studies described a rFIX (HIX) having better profile of post-translational modifications than rFIX produced by CHO cells. The aim of the study consisted to verify the improved post-translational modifications effect of HIX on in vivo recovery. HIX has been produced in a bioreactor and then purified from supernatants. In vitro activation and activity were evaluated measured by thrombin generation tests (TGT) and compared to commercial molecules, Benefix(®) , Mononine(®) . The three molecules were then administrated (i.v.) to FIX-knockout mice and two minutes after injection, blood samples were collected and subjected to human FIX-specific-ELISA and TGT. The clotting function of HIX, activation courses of HIX by FXIa and FVIIa-TF complex appear normal as did activation of Benefix(®) , Mononine(®) and TG constants of each FIX were equivalent. After injection to HB mice, circulating HIX did not present any significant difference in term of antigen value with Benefix(®) . Intriguingly, TGT were clearly exhibiting a better velocity for HIX than Benefix(®) and Mononine(®) . These data suggested that HIX may improve in vivo coagulant efficacy in comparison with the two commercial FIX injected at the same dose. The study shows that HuH-7-derived-rFIX has better in vivo haemostatic activity in hemophilia B mice compared to the reference rFIX molecule despite similar in vivo recovery rates, suggesting that HuH-7 cells could represent an effective cellular system for production of rFIX.

Influence of Imprinting of an X Chromosome and of the Methylene Tetrahydrofolate Reductase (MTHFR) 677C&gt;T Polymorphism on FVIII Activity

Background: In females, one X-chromosome is transcriptionally silenced by methylation of CpG sites in every cell during early embryogenesis. The methyl groups needed for DNA methylation are provided by the methylenetetrahydrofolate reductase (MTHFR). A polymorphism in the enzyme (677C>T, rs1801133) affects MTHFR concentrations and possibly also methylation of X-chromosomal genes. Whether X-chromosome inactivation (XCI) by methylation is associated with the MTHFR 677C>T genotype is currently unknown. It is also unclear whether the imprinting subsequently influences factor VIII activity in carrier females. Methods: We determined XCI in 61 hemophilia A carrier females and 174 control females by analysis of the human androgen receptor locus. Genotyping of MTHFR 677C>T was performed by mutagenically separated PCR, FVIII:C was tested by one-stage coagulation assays. Results: Slight skewing of XCI (X1:X2<1:3) was not associated with FVIII:C activity in hemophilia A carriers and controls. In contrast, strongly skewed XCI, which was observed in 10 carrier females, was associated with FVIII:C activity levels. If the X-chromosome carrying the mutated F8 gene was methylated, FVIII:C levels were higher. In contrast, FVIII:C levels were lower if the X-chromosome with the intact F8 gene was imprinted. We found a significant association of the 677TT MTHFR genotype with low FVIII in carrier females. The MTHFR genotype was not associated with FVIII:C in control females. Conclusion: Highly skewed imprinting of the X-chromosome influenced FVIII:C levels in carriers of hemophilia A. We found a significant association of MTHFR 677TT with low FVIII: C in hemophilia A carriers. Surprisingly, the MTHFR 677TT genotype was not significantly associated with X-chromosome inactivation. To elucidate the underlying mechanisms further studies will be necessary.

Pre-analytical issues in the measurement of circulating microparticles: current recommendations and pending questions

Pre-analytical issues in the measurement of circulating microparticles: current recommendations and pending questions

Poly-N-Acetylglucosamine Fibers Amplify the Effectiveness of Recombinant Factor VIIA on Clot Formation in Hemophilia B Canine Blood

Achieving hemostasis in anticoagulated patients is an increasingly important clinical issue. Poly-N-acetylglucosamine (pGlcNAc) nanofibers activate platelets by β3 subunit (CD61) and the von Willebrand receptor GP1b (CD42b) integrin signaling for generation of a prothrombotic surface membrane. Recombinant coagulation factor VIIa (rFVIIa) functions in hemophilia A and B by catalyzing formation of the Xa/Va complex on the surface of activated platelets. These observations suggest that pGlcNAc nanofibers may amplify the activity of rFVIIa in hemophilic blood. The activity of rFVIIa on platelets was tested by performing thromboelastographic analysis with blood from hemophilia B dogs in the presence of pGlcNAc nanofibers and increasing concentrations of rFVIIa. Mechanisms for hemostatic system activation were investigated with inhibitors of tissue factor, factor XIIa, and platelet function. Recombinant FVIIa was observed to partially restore the ability of the hemophiliac blood to form fibrin clots in a dose-dependent manner with thromboelastographic analysis. The addition of pGlcNAc nanofibers amplified the rFVIIa effect. The activity of rFVIIa and the amplification effect of pGlcNAc were dependent on platelet integrin function but independent of FXIIa and tissue factor activities. The pGlcNAc nanofibers amplify rFVIIa activity in hemophilia B canine blood by activating platelets through integrin-dependent mechanisms.

Repeated oral administration of chitosan/DNA nanoparticles delivers functional FVIII with the absence of antibodies in hemophilia A mice.

Current treatment of hemophilia A is expensive and involves regular infusions of factor (F)VIII concentrates. The supply of functional FVIII is further compromised by the generation of neutralizing antibodies. Thus, the development of an alternative safe, cost effective, non-invasive treatment that circumvents immune response induction is desirable. To evaluate the feasibility of oral administration of chitosan nanoparticles containing FVIII DNA to provide sustainable FVIII activity in hemophilia A mice. Nanoparticles were characterized for morphology, DNA protection and transfection efficiency. Oral administration of nanoparticles containing canine FVIII in C57Bl/6 FVIII(-/-) hemophilia A mice was evaluated for biodistribution, plasma FVIII activity and phenotypic correction. Sustainable FVIII expression was elucidated after repeated nanoparticle administration. Immune responses to repeated oral nanoparticle administration were also investigated. Chitosan nanoparticles had a particle size range of 200-400 nm and protected DNA from endonuclease and pH degradation. In addition, nanoparticles transfected HEK 293 cells resulted in expression of eGFP, luciferase and FVIII. Hemophilia A mice that ingested chitosan nanoparticles demonstrated transient canine FVIII expression reaching > 100 mU 1 day after treatment, together with partial phenotypic correction. The delivered FVIII plasmid DNA was detected in the intestine and, to a lesser extent, in the liver. Importantly, repeated weekly administrations restored FVIII activity. Furthermore, inhibitors and non-neutralizing FVIII antibodies were not detectable. Repeat oral administration of FVIII DNA formulated in chitosan nanoparticles resulted in sustained FVIII activity in hemophilic mice, and thus may provide a non-invasive alternative treatment for hemophilia A.

Targeting FVIII expression to endothelial cells regenerates a releasable pool of FVIII and restores hemostasis in a mouse model of hemophilia A

AbstractThe natural cell type(s) that synthesize and release factor VIII (FVIII) into the circulation are still not known with certainty. In vitro studies indicate that artificial expression of FVIII in endothelial cells produces an intracellular pool of FVIII that can be mobilized together with its carrier protein, von Willebrand factor (VWF), by agonists. Here, we show that expression of human B-domain deleted FVIII (hFVIII) in the vascular endothelium of otherwise FVIII-deficient mice results in costorage of FVIII and VWF in endothelial Weibel-Palade bodies and restores normal levels and activity of FVIII in plasma. Stored FVIII was mobilized into the circulation by subcutaneous administration of epinephrine. Human FVIII activity in plasma was strictly dependent on the presence of VWF. Endothelial-specific expression of hFVIII rescued the bleeding diathesis of hemophilic mice lacking endogenous FVIII. This hemostatic function of endothelial cell–derived hFVIII was suppressed in the presence of anti-FVIII inhibitory antibodies. These results suggest that targeting FVIII expression to endothelial cells may establish a releasable pool of FVIII and normalize plasma FVIII level and activity in hemophilia A, but does not prevent the inhibitory effect of anti-FVIII antibodies on the hemostatic function of transgene-derived hFVIII as is seen with platelet-derived FVIII expression.

A modified thrombin generation test for investigating very low levels of factor VIII activity in hemophilia A

Discrepancies between low levels of FVIII:C and clinical symptoms in severe hemophilia A are well-known. We have recently demonstrated that levels of FVIII:C < 0.2 IU/dl were consistent with clinical phenotype by clot waveform analysis, suggesting that precise measurement of very low levels of FVIII:C was clinically important. Thrombin generation tests (TGTs) triggered by tissue factor (TF) have been recently utilized to monitor coagulation function in hemophilia A. We examined whether TGT was useful for evaluating hemophilia A patients with very low levels of FVIII:C. TGTs in 40 hemophilia A plasmas with FVIII:C < 0.2-17 IU/dl (measured by clot waveform analysis using MDA-II) were performed using TF and/or ellagic acid (ELG). The lagtime in ELG-TGT at very low levels of FVIII:C was shortened dose-dependently, whilst this parameter in TF-TGT was not significantly affected. Other parameters (endogenous thrombin potential, peak thrombin, time to peak) correlated with FVIII:C levels to some extent in both assays (r = 0.4-0.7). Using a TF/ELG mixture in TGT, however, the correlation coefficients increased to ~0.85. TGT parameters correlated well with levels of FVIII:C > 0.2 IU/dl, although the lagtime was not especially informative. We conclude that modified TGT, using a TF/ELG mixture as the trigger, is useful for monitoring coagulation function at very low levels of FVIII:C in hemophilia A.

Factor VIII-Fc Fusion Protein Shows Extended Half-Life and Hemostatic Activity in Hemophilia A Dogs.

Abstract 545A recombinant B-domain-deleted factor VIII-Fc (rFVIIIFc) fusion protein was created as an approach to extend the half-life of FVIII. The pharmacokinetics and pharmacodynamics of rFVIIIFc were evaluated in the Chapel Hill colony of hemophilia A dogs. These dogs have a severe hemophilic phenotype comparable to the severe form of human disease with F.VIII < 1%. A single intravenous dose (125 IU/kg) was administered to four dogs and immediately corrected the clotting to normal as measured by whole blood clotting time (WBCT) and aPTT. The WBCT remained below 20 min, the time consistent with FVIII:C > 1%, through approximately 96 h. The range of WBCT in our normal dogs is 8 to 12 min. The concentration of rFVIIIFc in the plasma was measured by ELISA and the terminal half-life was 15.7 ± 1.7 hr. Similar results were obtained when rFVIIIFc was measured using a FVIII-specific chromogenic activity assay (half-life was 15.4 ± 0.3 hr). The concentration vs. time curves were similar using both methods. The activity data were converted to ng/mL using the specific activity of the test article that was used to dose the animals, and these data correlated well with the ELISA data, thus demonstrating that the protein that was measured by ELISA was fully active.Two of the dogs also received a single dose of recombinant B-domain deleted FVIII (rBDD-FVIII, ReFacto®), 114 IU/kg for one dog and 120 IU/kg rBDD-FVIII for the other, and then received rFVIIIFc (125 IU/kg) 72 hr later in a cross over design. Clotting was corrected to normal immediately after dosing with both rBDD-FVIII and rFVIIIFc (determined by WBCT and clotting activity measured using an aPTT assay). However, the WBCT normalization after rFVIIIFc lasted for approximately twice as long compared to rBDD-FVIII and the half-lives determined from the ELISA data for FVIIIFc (15.7 ± 1.7 hr) were twice those determined for rBDD-FVIII (7.0 hr and 6.7 hr). No adverse clinical signs were detected with any of the infusions. Therefore construction of an Fc fusion of FVIII produces a molecule with a defined mechanism of action that has an increased half life and the potential to provide prolonged protection from bleeding. Disclosures:Dumont:Biogen Idec (Syntonix Subsidiary): Employment. Kamphaus:Biogen Idec (Syntonix Subsidiary): Employment. Fraley:Biogen Idec/Syntonix Subsidiary: Employment. Ashworth:Biogen Idec (Syntonix Subsidiary): Employment. Bitonti:Biogen Idec/Syntonix Subsidiary: Employment.

Directed Engineering of a High-expression Chimeric Transgene as a Strategy for Gene Therapy of Hemophilia A

Human coagulation factor VIII (fVIII) is inefficiently biosynthesized in vitro and has proven difficult to express at therapeutic levels using available clinical gene-transfer technologies. Recently, we showed that a porcine and certain hybrid human/porcine fVIII transgenes demonstrate up to 100-fold greater expression than human fVIII. In this study, we extend these results to describe the use of a humanized, high-expression, hybrid human/porcine fVIII transgene that is 89% identical to human fVIII and was delivered by lentiviral vectors (LVs) to hematopoietic stem cells for gene therapy of hemophilia A. Recombinant human immunodeficiency virus-based vectors encoding the fVIII chimera efficiently transduced human embryonic kidney (HEK)-293T cells. Cells transduced with hybrid human/porcine fVIII encoding vectors expressed fVIII at levels 6- to 100-fold greater than cells transduced with vectors encoding human fVIII. Transplantation of transduced hematopoietic stem and progenitor cells into hemophilia A mice resulted in long-term fVIII expression at therapeutic levels despite <5% genetically modified blood mononuclear cells. Furthermore, the simian immunodeficiency virus (SIV) -derived vector effectively transduced the human hematopoietic cell lines K562, EU1, U.937, and Jurkat as well as the nonhematopoietic cell lines, HEK-293T and HeLa. All cell lines expressed hybrid human/porcine fVIII, albeit at varying levels with the K562 cells expressing the highest level of the hematopoietic cell lines. From these studies, we conclude that humanized high-expression hybrid fVIII transgenes can be utilized in gene therapy applications for hemophilia A to significantly increase fVIII expression levels compared to what has been previously achieved.

Regression analysis of amino acid substitutions and factor IX activity in hemophilia B

Hemophilia B is caused by a deficit or decreased activity of factor IX. Mutation in factor IX is made up of a majority of amino acid substitutions. We examined the relation between the activation level of factor IX and the category of amino acid substitution with regression analysis. As parameters, we used four physical-chemical parameters of amino acids and Dayhoff’s PAM matrix. In addition, the average of the activation level with the same amino acid substitution was used for the analysis. We analyzed the relationship between variations containing cysteine or factor IX’s seven regions and activity levels.

Hematopoietic Stem Cells Encoding Porcine Factor VIII Induce Pro-coagulant Activity in Hemophilia A Mice with Pre-existing Factor VIII Immunity

The development of inhibitory antibodies directed against factor VIII (fVIII) remains the most significant clinical complication associated with the treatment of hemophilia A. Recently, we demonstrated that transplantation of genetically modified hematopoietic stem cells containing a high-expression porcine fVIII transgene promoted sustained high-level fVIII expression in naïve hemophilia A mice. In the current study, a similar gene transfer strategy was tested in hemophilia A mice harboring clinically significant anti-human factor VIII (anti-hfVIII) inhibitory antibody titers. Although the majority of mice contained circulating antibodies that cross-reacted with and inhibited porcine fVIII activity, transplantation of genetically modified hematopoietic stem cells containing a porcine fVIII transgene into myeloablated hemophilia A mice induced high-level fVIII activity. Furthermore, anti-hfVIII antibody titers steadily declined throughout the course of the study. However, non-myeloablative transplantation conditioning resulted in only partial success. No correlation between pre-transplantation antibody titers and post-transplantation fVIII activity levels or donor cell engraftment was observed. These data suggest that hematopoietic stem cell transplantation-based gene therapy incorporating a high-expression porcine fVIII transgene can be utilized successfully to treat hemophilia A patients harboring anti-hfVIII inhibitors.

Hematopoietic Stem Cells Encoding Porcine Factor VIII Induce Pro-coagulant Activity in Hemophilia A Mice with Pre-existing Factor VIII Immunity

Hematopoietic Stem Cells Encoding Porcine Factor VIII Induce Pro-coagulant Activity in Hemophilia A Mice with Pre-existing Factor VIII Immunity

Multiyear therapeutic benefit of AAV serotypes 2, 6, and 8 delivering factor VIII to hemophilia A mice and dogs

Hemophilia A, a deficiency of functional coagulation factor VIII (FVIII), is treated via protein replacement therapy. Restoring 1% to 5% of normal blood FVIII activity prevents spontaneous bleeding, making the disease an attractive gene therapy target. Previously, we have demonstrated short-term activity of a liver-specific AAV2 vector expressing canine B-domain-deleted FVIII (cFVIII) in a hemophilia canine model. Here, we report the long-term efficacy and safety of AAV-cFVIII vectors of serotypes 2, 5, 6, and 8 in both hemophilia A mice and dogs. AAV6-cFVIII and AAV8-cFVIII restored physiologic levels of plasma FVIII activity in hemophilia A mice. The improved efficacy is attributed to more efficient gene transfer in liver compared with AAV2 and AAV5. However, supraphysiologic cFVIII levels correlated with the formation of cFVIII-neutralizing antibodies in these mice. Of importance, hemophilia A dogs that received AAV2-cFVIII, AAV6-cFVIII, and AAV8-cFVIII have persistently expressed therapeutic levels of FVIII, without antibody formation or other toxicities, for more than 3 years. However, liver transduction efficiencies are similar between AAV2, AAV6, and AAV8 serotypes in hemophilia A dogs, in contrast to mice. In summary, this is the first report demonstrating multiyear therapeutic efficacy and safety of multiple AAV-cFVIII vectors in hemophilia A dogs and provides the basis for human clinical studies.

Lower Inhibitor Development in Hemophilia A Mice following Administration of Recombinant Factor VIII-O-Phospho-L-serine Complex

Factor VIII is a multidomain protein composed of A1, A2, B, A3, C1, and C2 domains. Deficiency or dysfunction of factor VIII causes hemophilia A, a bleeding disorder. Administration of exogenous recombinant factor VIII as a replacement leads to development of inhibitory antibodies against factor VIII in 15-30% of hemophilia A patients. Hence, less immunogenic preparations of factor VIII are highly desirable. Inhibitory antibodies against factor VIII are mainly directed against immunodominant epitopes in C2, A3, and A2 domains. Further, several universal epitopes for CD4+ T-cells have been identified within the C2 domain. The C2 domain is also known to interact specifically with phosphatidylserine-rich lipid vesicles. Here, we have investigated the hypothesis that complexation of O-phospho-l-serine, the head group of phosphatidylserine, with the C2 domain can reduce the overall immunogenicity of factor VIII. The biophysical (circular dichroism and fluorescence) and biochemical studies (ELISA and size exclusion chromatography) showed that O-phospho-l-serine binds to the phospholipid-binding region in the C2 domain, and this interaction causes subtle changes in the tertiary structure of the protein. O-Phospho-l-serine also prevented aggregation of the protein under thermal stress. The immunogenicity of the factor VIII-O-phospho-l-serine complex was evaluated in hemophilia A mice. The total and inhibitory antibody titers were lower for factor VIII-O-phospho-l-serine complex compared with factor VIII alone. Moreover, factor VIII administered as a complex with O-phospho-l-serine retained in vivo activity in hemophilia A mice. Our results suggest that factor VIII-O-phospho-l-serine complex may be beneficial to increase the physical stability and reduce immunogenicity of recombinant factor VIII preparations.