BACKGROUND Hemophilia B, an X-linked disorder, is ideally suited for gene therapy. We investigated the use of a new gene therapy in patients with the disorder. METHODS We infused a single dose of a serotype-8–pseudotyped, self-complementary adenovirus-associated virus (AAV) vector expressing a codon-optimized human factor IX (FIX) transgene (scAAV2/8-LP1-hFIXco) in a peripheral vein in six patients with severe hemophilia B (FIX activity, <1% of normal values). Study participants were enrolled sequentially in one of three cohorts (given a high, intermediate, or low dose of vector), with two participants in each group. Vector was administered without immunosuppressive therapy, and participants were followed for 6 to 16 months. RESULTS AAV-mediated expression of FIX at 2 to 11% of normal levels was observed in all participants. Four of the six discontinued FIX prophylaxis and remained free of spontaneous hemorrhage; in the other two, the interval between prophylactic injections was increased. Of the two participants who received the high dose of vector, one had a transient, asymptomatic elevation of serum aminotransferase levels, which was associated with the detection of AAV8-capsid–specific T cells in the peripheral blood; the other had a slight increase in liver-enzyme levels, the cause of which was less clear. Each of these two participants received a short course of glucocorticoid therapy, which rapidly normalized aminotransferase levels and maintained FIX levels in the range of 3 to 11% of normal values. CONCLUSIONS Peripheral-vein infusion of scAAV2/8-LP1-hFIXco resulted in FIX transgene expression at levels sufficient to improve the bleeding phenotype, with few side effects. Although immune-mediated clearance of AAV-transduced hepatocytes remains a concern, this process may be controlled with a short course of glucocorticoids without loss of transgene expression. (Funded by the Medical Research Council and others; ClinicalTrials.gov number, NCT00979238.)

BACKGROUND In patients with severe hemophilia B, gene therapy that is mediated by a novel self-complementary adeno-associated virus serotype 8 (AAV8) vector has been shown to raise factor IX levels for periods of up to 16 months. We wanted to determine the durability of transgene expression, the vector dose–response relationship, and the level of persistent or late toxicity. METHODS We evaluated the stability of transgene expression and long-term safety in 10 patients with severe hemophilia B: 6 patients who had been enrolled in an initial phase 1 dose-escalation trial, with 2 patients each receiving a low, intermediate, or high dose, and 4 additional patients who received the high dose (2×1012 vector genomes per kilogram of body weight). The patients subsequently underwent extensive clinical and laboratory monitoring. RESULTS A single intravenous infusion of vector in all 10 patients with severe hemophilia B resulted in a dose-dependent increase in circulating factor IX to a level that was 1 to 6% of the normal value over a median period of 3.2 years, with observation ongoing. In the high-dose group, a consistent increase in the factor IX level to a mean (±SD) of 5.1±1.7% was observed in all 6 patients, which resulted in a reduction of more than 90% in both bleeding episodes and the use of prophylactic factor IX concentrate. A transient increase in the mean alanine aminotransferase level to 86 IU per liter (range, 36 to 202) occurred between week 7 and week 10 in 4 of the 6 patients in the high-dose group but resolved over a median of 5 days (range, 2 to 35) after prednisolone treatment. CONCLUSIONS In 10 patients with severe hemophilia B, the infusion of a single dose of AAV8 vector resulted in long-term therapeutic factor IX expression associated with clinical improvement. With a follow-up period of up to 3 years, no late toxic effects from the therapy were reported. (Funded by the National Heart, Lung, and Blood Institute and others; ClinicalTrials.gov number, NCT00979238.)

To cite this article: Gatt A, Riddell A, Calvaruso V, Tuddenham EG, Makris M, Burroughs AK. Enhanced thrombin generation in patients with cirrhosis-induced coagulopathy. J Thromb Haemost 2010; 8: 1994-2000 Summary. Background: Prothrombin time (PT) and the international normalized ratio (INR) are still routinely measured in patients with liver cirrhosis to ÔassessÕ their bleeding risk despite the lack of correlation with the two. Thrombin generation (TG) assays are global assays of coagulation that are showing promise in assessing bleeding and thrombosis risks. Aim: To study the relationship between the INR and TG profiles in cirrhosis-induced coagulopathy. Methods: Seventy-three patients with cirrhosis were studied. All TG parameters were compared with those from a normal control group. Contact activation was prevented using corn trypsin inhibitor. TG was also assayed in the presence of Protac Ò . The endogenous thrombin potential (ETP) ratio was derived by dividing the ETP with ProtacÒ by the ETP without ProtacÒ. Results: The INR (mean 1.7) did not correlate with the ETP and the velocity of TG (P > 0.05). There was no difference between the lag time and ETP of the two groups (P > 0.05). The velocity of TG was increased in cirrhosis (67.95 ± 34.8 vs. 45.05 ± 25.9 nM min )1 ; P = 0.016) especially in patients with INRs between 1.21 and 2.0. Both the ETP with Protac Ò and the ETP ratio were increased in cirrhosis (mean 1074 ± 461.4 vs. 818 ± 357.9 nM min, P = 0.004 and 0.80 ± 0.21 vs. 0.44 ± 0.15, P £ 0.0001, respectively). Conclusion: Despite a raised INR, TG parameters are consistent with a hypercoagulable profile in cirrhosis-related coagulopathy. This confirms that the PT or INR should not be used to assess bleeding risk in these patients, and other parameters, such as TG, need to be explored as clinical markers of coagulopathy.

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Emicizumab is a bispecific antibody that activates FX to FXa in the absence of FVIII. It has been shown to reduce bleeding episodes in people with haemophilia A complicated by a FVIII inhibitor. Despite the protection against bleeds, some breakthrough bleeds are inevitable and these may require additional haemostatic treatment. Emicizumab has been associated with severe adverse events when co-administered with activated prothrombin complex concentrate. To minimize the risk of adverse events, the UK Haemophilia Centre Doctors' Organisation issues the following updated interim guidance to its Inhibitor Guidelines for managing patients receiving Emicizumab based on the limit published information available in February 2018.

Background: We have previously shown that a single intravenous administration of a self-complementary adeno-associated virus (scAAV) vector containing a codon-optimised factor IX gene, under control of a synthetic liver specific promoter and pseudotyped with serotype 8 capsid, (scAAV2/8-LP1-hFIXco) resulted in a dose-dependent increase in plasma FIX levels in all 10 enrolled severe hemophilia B (HB) patients (ClinicalTrials.gov:NCT00979238; Nathwani et al 2011). FIX activity was stably maintained for at least 3 years (Nathwani et al 2014) but concerns over FIX expression declining over time remain. This is because AAV-mediated transgene expression is mediated mainly by episomally retained viral genomes, which may be lost with natural hepatocyte turn-over. The only vector-associated adverse event was an asymptomatic rise in liver enzymes associated with a decline in FIX levels, occurring within 3 months of gene transfer in two-thirds of the patients treated at a dose of 2x1012 vector genomes(vg)/kg. Liver enzymes normalized with corticosteroids without complete loss of transgene expression. There was no long-lasting toxicity over a period of 3 years but further follow-up is required. The vector preparation used contained an excess of empty capsids, which lacked a full-length viral genome, and are therefore, non-functional but capable of provoking an immune response against transduced hepatocytes. Therefore, a new clinical preparation of scAAV2/8-LP1-hFIXco was manufactured from which most of the empty particles were removed by caesium chloride density centrifugation in the hope that this would reduce the risk of hepatotoxicity. We report on the evaluation of this new vector preparation in severe HB patients and provide an update on up to 8 years follow-up of our original cohort of patients. Methods: Ten subjects were recruited in 2010-2012 to the initial dose-escalation/extension study arm, which entailed a single intravenous infusion of scAAV2/8-LP1-hFIXco (full: empty capsid ratio ~1:10) at a dose of either 2x1011vg/kg, 6x1011vg/kg or 2x1012vg/kg. Two severe HB patients (FIX <1%) were enrolled into the first and mid-dose cohorts, with six patients treated at the high dose. In a follow-on study arm, two severe HB subjects received a dose of 2x1012vg/kg of the new scAAV2/8-LP1-hFIXco preparation (full: empty capsid ratio 1:3) whilst the next 2 patients were treated at a dose of 5x1012vg/kg. In both arms, vector was administered without prophylactic immunosuppression but corticosteroids (starting at 60mg/day) were commenced if liver enzymes increased to ≥2 fold over baseline levels after gene transfer. Results: Transgenic FIX activity levels have remained stable in all 10 subjects treated in the initial dose escalation/extension arm over a median follow-up of 6.7±1.0 years with mean levels in the three dose cohorts at the time of reporting of 1.9±0.6, 2.3±0.3 and 5.1±1.4 IU/l respectively. Over this period, annual FIX concentrate usage has dropped by 66% and annual bleed rate has declined by 82% when compared to pre-gene therapy levels. No late toxicity was observed. Neutralising antibodies to FIX were not detected in any patient but anti-AAV8 capsid-specific antibody levels persisted at high titres in 9 of 10 patients. In patients treated with the new preparation of scAAV2/8-LP1-hFIXco (median follow up = 2.1±1.4 years), mean FIX activity in the 2x1012vg/kg dose cohort was 2.6±0.7 IU/l. This is lower than observed previously at this dose level, but the difference is not statistically significant. Mean steady state FIX levels in the 5x1012vg/kg cohort were 17±5 IU/l. FIX antigen to activity ratio was 1:1. Elevation of serum alanine aminotransferase was observed in 3 of 4 patients treated with the new vector preparation, requiring treatment with corticosteroids. Conclusion: This is the first report to demonstrate stable therapeutic expression of FIX in patients with severe HB over a period of 8 years following systemic administration of scAAV2/8-LP1-hFIXco without late toxicities. We show for the first time that reducing the capsid load by removing empty AAV capsids does not appear to reduce the incidence of hepatotoxicity in patients with severe HB suggesting that other factors are involved in the aetiology of this process. Disclosures Nathwani: Freeline: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties; BioMarin: Consultancy, Patents & Royalties; UniQure: Patents & Royalties. Tuddenham:BioMarin: Consultancy, Patents & Royalties; Freeline: Consultancy. Chowdary:Freeline: Consultancy; Baxalta (Shire): Honoraria, Membership on an entity's Board of Directors or advisory committees; Bayer: Honoraria; Pfizer: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Biogen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Novo Nordisk: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; CSL Behring: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Swedish Orphan Biovitrum AB (Sobi): Honoraria. McIntosh:Freeline: Consultancy. Recht:Biogen: Research Funding; Shire: Research Funding; Novo Nordisk: Membership on an entity's Board of Directors or advisory committees, Research Funding; Kedrion: Membership on an entity's Board of Directors or advisory committees.

Investigation of 3 families with bleeding symptoms demonstrated a defect in the collagen-binding activity of von Willebrand factor (VWF) in association with a normal VWF multimeric pattern. Genetic analysis showed affected persons to be heterozygous for mutations in the A3 domain of VWF: S1731T, W1745C, and S1783A. One person showed compound heterozygosity for W1745C and R760H. W1745C and S1783A have not been reported previously. The mutations were reproduced by site-directed mutagenesis and mutant VWF expressed in HEK293T cells. Collagen-binding activity measured by immunosorbent assay varied according to collagen type: W1745C and S1783A were associated with a pronounced binding defect to both type I and type III collagen, whereas the principal abnormality in S1731T patients was a reduction in binding to type I collagen only. The multimer pattern and distribution of mutant proteins were indistinguishable from wildtype recombinant VWF, confirming that the defect in collagen binding resulted from the loss of affinity at the binding site and not impairment of high-molecularweight multimer formation. Our findings demonstrate that mutations causing an abnormality in the binding of VWF to collagen may contribute to clinically significant bleeding symptoms. We propose that iso- Introductionvon Willebrand factor (VWF) is a large multimeric glycoprotein that has 2 important roles in hemostasis. These are stabilizing factor VIII (FVIII), by acting as its carrier protein in the circulation, and attaching activated platelets to the subendothelium via binding to the GpIb receptor on platelets and to collagen in the subendothelial matrix. Enhancement of the platelet-subendothelium interaction is vital at sites of vascular injury during conditions of high shear stress. VWF binds to collagen via 2 sites: the A3 domain (residues 1683-1874) contains the main site for fibrillar collagen types I and III found within perivascular connective tissue, 1,2 and a second site in the A1 domain (residues 1260-1471) binds nonfibrillar collagen type VI within the subendothelial matrix. 3,4 However, the A1 domain can also bind collagen types I and III, and the relative importance of the 2 domains has been extensively but inconclusively investigated. 5,6 In all cases, it appears that affinity of VWF for collagen is heavily dependent on the presence of VWF multimers of high or ultrahigh molecular weight. 7 Type 2 von Willebrand disease (VWD) is characterized by a qualitative defect in VWF and is diagnosed by demonstration of a discrepancy between circulating plasma levels of VWF and its functional activity. In addition to measuring GpIb␣-dependent function using ristocetin cofactor activity (VWF:RCo), it has been recommended that collagen-binding activity (VWF:CB) is analyzed in the subclassification of type 2 VWD. 8 A single defect in VWF collagen binding in association with normal multimeric structure has previously been reported. 9 In this report, we describe the phenotypic and genotypic characterization of 3 families with bleeding symptoms...

AbstractWe have examined the effect of the O-linked glycan (OLG) structures of VWF on its interaction with the platelet receptor glycoprotein Ibα. The 10 OLGs were mutated individually and as clusters (Clus) on either and both sides of the A1 domain: Clus1 (N-terminal side), Clus2 (C-terminal side), and double cluster (DC), in both full-length-VWF and in a VWF construct spanning D′ to A3 domains. Mutations did not alter VWF secretion by HEK293T cells, multimeric structure, or static collagen binding. The T1255A, Clus1, and DC variants caused increased ristocetin-mediated GPIbα binding to VWF. Platelet translocation rate on OLG mutants was increased because of reduced numbers of GPIbα binding sites but without effect on bond lifetime. In contrast, OLG mutants mediated increased platelet capture on collagen under high shear stress that was associated with increased adhesion of these variants to the collagen under flow. These findings suggest that removal of OLGs increases the flexibility of the hinge linker region between the D3 and A1 domain, facilitating VWF unfolding by shear stress, thereby enhancing its ability to bind collagen and capture platelets. These data demonstrate an important functional role of VWF OLGs under shear stress conditions.