Transduction with recombinant adenoassociated virus (AAV) vectors is limited by the need to convert its single-stranded (ss) genome to transcriptionally active double-stranded (ds) forms. For AAVmediated hemophilia B (HB) gene therapy, we have overcome this obstacle by constructing a liver-restricted mini-human factor IX (hFIX) expression cassette that can be packaged as complementary dimers within individual AAV particles. Molecular analysis of murine liver transduced with these self-complementary (sc) vectors demonstrated rapid formation of active ds-linear genomes that persisted stably as concatamers or monomeric circles. This unique property resulted in a 20-fold improvement in hFIX expression in mice over comparable ssAAV vectors. Administration of only 1 ؋ 10 10 scAAV particles led to expression of hFIX at supraphysiologic levels (8I U/mL) and correction of the bleeding diathesis in FIX knock-out mice. Of importance, therapeutic levels of hFIX (3%-30% of normal) were achieved in nonhuman primates using a significantly lower dose of scAAV than required with ssAAV. Furthermore, AAV5-pseudotyped scAAV vectors mediated successful transduction in macaques with pre-existing immunity to AAV8. Hence, this novel vector represents an important advance for hemophilia B gene therapy. IntroductionThe liver is an important target for gene therapy of a variety of genetic disorders, one of which is hemophilia B (HB), a lifethreatening bleeding disorder that arises from mutations in the blood coagulation factor IX (FIX) gene. By maintaining plasma FIX levels above 1% of normal (Ͼ 0.05 g/mL), the incidence of spontaneous hemorrhage is dramatically reduced and so the therapeutic end point for HB gene therapy is modest. 1 Currently, adeno-associated virus (AAV) vectors are the most promising for HB gene therapy and have been the focus of 2 recent clinical trials. 2 Efficient transduction with AAV is, however, limited by the need to convert its single-stranded (ss) genome into transcriptionally active double-stranded (ds) forms in target cells because of its dependence on host-cell-mediated DNA synthesis of the leading strand 3 or annealing of complementary genomes derived from separate virions. 4 Coinfection with adenovirus 5 or priming the target tissues with genotoxic agents 6,7 can enhance ds transgene formation, but the clinical use of these approaches is limited by potential toxicity. Rapid uncoating of the viral genome, as recently described with AAV8 vectors, allows efficient annealing of the ssAAV provirus to form double-stranded genomes. This unique biologic property is responsible for the 10-to 100-fold higher transduction of the liver with rAAV8 when compared with AAV2 vectors in murine models. [8][9][10] Even so, almost 10 13 AAV8 vector particles are required to achieve 100% hepatocyte transduction in mice, a level that is required for successful gene therapy of some metabolic disorders of the liver. 11 This high vector dose is problematic because it may (1) Supported by The Assisi Foundation of Memphis; the Ame...

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Since 1996 the HAMSTeRS (Haemophilia A Mutation, Search, Test and Resource Site) WWW site has provided an online resource for access to data on the molecular pathology of haemophilia A, replacing previous text editions of the Haemophilia A Database published in Nucleic Acids Research . This report describes the continued development of the site (version 4), and in particular the expansion of factor VIII (FVIII) structure-related features. Access to the mutation database itself, both for searching the listings and for submission of new mutations, is via custom-designed forms: more powerful Boolean searches of the point mutations in the database are also available. During 1997 a total of 22 novel missense mutations were reported, increasing the total number of unique variants now described to 252 (238 in exonic sequences and 14 at intronic splice junctions). Currently, a total of 586 individual reports with associated phenotypic data are available for searching by any category including phenotype. The FVIII structure section now includes a download of a FVIII A domain homology model in Protein Data Bank format and a multiple alignment of the FVIII amino-acid sequencies from four species (human, murine, porcine and canine) in addition to the virtual reality simulations, secondary structural data and FVIII animation already available. Finally, to aid navigation across this site, a clickable roadmap of the main features provides easy access to the page desired. Our intention is that continued development and updating of the site shall provide workers in the fields of molecular and structural biology with a one-stop resource site to facilitate FVIII research and education. The HAMSTeRS URL is http://europium.mrc.rpms.ac.uk

lation factor IX provides novel insights into the phenotypes and genetics of hemophilia B. J Thromb Haemost 2013; 11: 1329-40.Summary. Background: Factor IX (FIX) is important in the coagulation cascade, being activated to FIXa on cleavage. Defects in the human F9 gene frequently lead to hemophilia B. Objective: To assess 1113 unique F9 mutations corresponding to 3721 patient entries in a new and up-to-date interactive web database alongside the FIXa protein structure. Methods: The mutations database was built using MySQL and structural analyses were based on a homology model for the human FIXa structure based on closely-related crystal structures. Results: Mutations have been found in 336 (73%) out of 461 residues in FIX. There were 812 unique point mutations, 182 deletions, 54 polymorphisms, 39 insertions and 26 others that together comprise a total of 1113 unique variants. The 64 unique mild severity mutations in the mature protein with known circulating protein phenotypes include 15 (23%) quantitative type I mutations and 41 (64%) predominantly qualitative type II mutations. Inhibitors were described in 59 reports (1.6%) corresponding to 25 unique mutations. Conclusion: The interactive database provides insights into mechanisms of hemophilia B. Type II mutations are deduced to disrupt predominantly those structural regions involved with functional interactions. The interactive features of the database will assist in making judgments about patient management.

Despite recent studies, the organization of coagulation factor VIII (FVIII) on a phospholipid (PL) membrane is not known in detail. Thus, 2-dimensional (2D) crystals of human FVIII lacking the B domain were prepared for electron microscopy onto negatively charged PL monolayers. The 3-dimensional (3D) density map of the PL-bound FVIII protein was calculated at 1.5 nm. Existing atomic data and models for FVIII domains were fitted unambiguously within the 3D density map of the molecule. FVIII domains arrangement followed a compact spiral organization with the A3 domains in close association with the C1 and C2 domains near the PL surface. Viewed toward the membrane the A domains' heterotrimer is oriented side-on with the pseudo-3-fold axis almost parallel to the PL surface and A1 fully covering C1. The C2 domain is partially overlapped by the A2 domain of an adjacent molecule in the 2D crystal, favoring close packing. Viewed parallel to the membrane, C2 is slightly inclined to the PL surface covering an area of 12 nm 2 . Four C2 loops are embedded within the lipid monolayer at about 0.7 to 1.0 nm depth. C1 forms almost a right angle with C2, its long axis nearly parallel to the membrane. The proposed structure for membrane-bound FVIII results from modeling of the FVIII domains within a 3D density map obtained from electron crystallography and accords with the main biochemical and structural information known to date. A model is proposed for FVIIIa and factor IXa assembly within the membrane-bound factor X-activating complex. IntroductionFactor VIII (FVIII) is a plasma protein essential for blood coagulation and is deficient or defective in individuals with hemophilia A. FVIII is synthesized as a 300-kd precursor protein with domain structure A1-A2-B-A3-C1-C2. 1,2 In plasma it circulates as a series of noncovalently bound heterodimers, produced by proteolytic cleavage at the B-A3 junction or within the B domain. 3 Thus, FVIII in vivo consists of a heavy chain (HC) of variable molecular mass (from 90 kd, when only the A1-A2 domains are present, to 220 kd when the full-length B domain is present) and a light chain (A3-C1-C2; LC) of 76 kd molecular mass (for a review, see Lenting et al 4 ).In the process of blood coagulation, FVIII acts as a cofactor to the serine protease factor IXa (FIXa). Before the initiation of coagulation, FVIII circulates in a noncovalent complex with von Willebrand factor (VWF). Limited proteolysis near the N-terminus of LC by traces of thrombin removes a peptide containing a VWF-binding site, resulting in release of free FVIII. Further proteolysis between the A1 and A2 domains results in generation of FVIIIa, the active heterotrimeric cofactor. Binding of FVIIIa to negatively charged phospholipid (PL) and to factor IXa (FIXa) form the factor X-ase (FX-ase) complex. The product of the reaction, activated factor X (FXa), forms with activated factor V (cofactor FVa) a similar membrane-bound system, the prothrombinase complex that converts prothrombin to thrombin. 6 The serine proteases FIXa and F...

SummaryA recent report from the Leiden Thrombophilia Survey identified high factor VIII activity levels as an independent risk factor for venous thromboembolism in a population survey. As the study measure for factor VIII was a one-stage coagulation assay, and since markers for the acute phase reaction were not assessed, it remained uncertain whether the increase was due to a constitutional increased rate of synthesis, to circulating activated factor VIII, or to an acute phase response.We added factor VIII activity assay (FVIII:C), factor VIII antigen (FVIILAg), vWF antigen (vWF:Ag), ABO blood group, fibrinogen and C-reactive protein to our routine thrombophilia screen of patients referred because of unexplained thromboembolism.Elevated FVIILC (>1.5 iu/ml) emerged as the single commonest abnormality detected in 25.4% of a group of 260 such patients.FVIILC and FVIILAg were highly correlated (p = 0.003), showing that this represented a true increase in FVIII. In 4 of 46 patients this was clearly attributable to an acute phase reaction. Eleven others showed minor elevation of ESR and one of CRP. Neither FVIILC or FVIII:Ag showed significant correlation with fibrinogen, ESR or C-reactive protein by non parametric analysis.Although there was an excess of patients with B blood group (known to be associated with FVIILC levels which are -15% higher than those in blood group 0), this could not account for the marked elevation of factor VIII observed in these patients.We conclude that factor VIII activity assay should be a routine part of thrombophilia screening. We are investigating the cause of the increased synthesis, initially by means of family studies and linkage analysis with polymorphic markers of the FVIII locus. We postulate that it may be constitutive in some cases and in others an abnormal or exaggerated response to inflammatory stimuli.

The hemophilia A mutation database lists more than 160 missense mutations: each represents a molecular defect in the FVIII molecule, resulting in the X-linked bleeding disorder hemophilia A with a clinical presentation varying from mild to severe. Without a three-dimensional FVIII structure it is in most cases impossible to explain biological dysfunction in terms of the underlying molecular pathology. However, recently the crystal structure of the homologous human plasma copper-binding protein ceruloplasmin (hCp) has been solved, and the A domains of FVIII share approximately 34% sequence identity with hCp. This advance has enabled the building of a molecular model of the A domains of FVIII based on the sequence identity between the two proteins. The model allows exploration of predictions regarding the general features of the FVIII molecule, such as the binding-sites for factor IXa and activated protein C; it has also allowed the mapping of more than 30 selected mutations with known phenotype from the database, and the prediction of hypothetical links to dysfunction in all but a few cases. A computer-generated molecular model such as that reported here cannot substitute for a crystal structure. However, until such a structure for FVIII becomes available, the model represents a significant advance in modeling FVIII; it should prove a useful tool for exploiting the increasing amount of information in the hemophilia A mutation database, and for selecting appropriate targets for investigation of the structure-function relationships via mutagenesis and expression in vitro.

Factor VII (FVII) is a zymogen for a vitamin K‐dependent serine protease essential for the initiation of blood coagulation. It is synthesized primarily in the liver and circulates in plasma at a concentration of approximately 0.5 μg/ml (10 nmol/L). The FVII gene (F7) is located on chromosome 13 (13q34), consists of 9 exons, and spans approximately 12kb. It encodes a mature protein of 406 amino acids, which has an N‐terminal domain (Gla) post‐translationally modified by γ‐carboxylation of glutamic acid residues, two domains with homology to epidermal growth factor (EGF1 and 2), and a C‐terminal serine protease domain. The single chain zymogen is activated by proteolytic cleavage at Arg152‐Ile153. There are 238 individuals described in the world literature with mutations in their F7 genes (FVII mutation database; europium.csc.mrc.ac.uk). Complete absence of FVII activity in plasma is usually incompatible with life, and individuals die shortly after birth due to severe hemorrhage. The majority of individuals with mutations in their F7 gene(s), however, are either asymptomatic or the clinical phenotype is unknown. In general, a severe bleeding phenotype is only observed in individuals homozygous for a mutation in their F7 genes with FVII activities (FVII:C) below 2% of normal, however, a considerable proportion of individuals with a mild‐moderate bleeding phenotype have similar FVII:C by in vitro assay. The failure of in vitro tests to differentiate between these groups may be due to lack of sensitivity in the assays to the very low amounts of FVII:C, which are sufficient to initiate coagulation in vivo. A number of polymorphisms have been identified in the F7 gene and some have been shown to influence plasma FVII antigen levels. Hum Mutat 17:3–17, 2001. © 2001 Wiley‐Liss, Inc.