Transgene-host cell interactions mediate significant influences on the production, stability, and function of recombinant canine FVIII.

Recombinant FVIII manufacturing is characterized by poor product stability and low yields. Codon-optimization of transgenes accelerates translation by exploiting the synonymous codon usage bias of a species. However, this can alter the performance of the final product.

Analysis of the role of von Willebrand factor, platelet glycoprotein VI-, and α2β1-mediated collagen binding in thrombus formation.

Rare missense mutations in the von Willebrand factor (VWF) A3 domain that disrupt collagen binding have been found in patients with a mild bleeding phenotype. However, the analysis of these aberrant VWF-collagen interactions has been limited. Here, we have developed mouse models of collagen-binding mutants and analyzed the function of the A3 domain using comprehensive in vitro and in vivo approaches.

Omental implantation of BOECs in hemophilia dogs results in circulating FVIII antigen and a complex immune response.

Ex vivo gene therapy strategies avoid systemic delivery of viruses thereby mitigating the risk of vector-associated immunogenicity. Previously, we delivered autologous factor VIII (FVIII)-expressing blood outgrowth endothelial cells (BOECs) to hemophilia A mice and showed that these cells remained sequestered within the implanted matrix and provided therapeutic levels of FVIII. Prior to translating this strategy into the canine (c) model of hemophilia A, we increased cFVIII transgene expression by at least 100-fold with the use of the elongation factor 1 alpha (EF1α) promoter and a strong endothelial enhancer element.

Quantitative and qualitative changes of von Willebrand factor and their impact on mortality in patients with end-stage kidney disease.

von Willebrand factor (vWF) antigen levels are elevated in patients with end-stage kidney disease (ESKD). We determined the quantitative and qualitative abnormalities of vWF and factors influencing vWF proteolysis in participants with ESKD compared with age-matched controls and determined the association between abnormalities in vWF and mortality over 4 years of follow-up. vWF : Ag and von Willebrand factor propeptide (vWFpp) levels, vWF functional activity (vWF :RCo), vWF multimer profiles, ADAMTS-13, thrombospondin 1 (TSP-1), and interleukin 6 (IL-6) were evaluated before and after a single hemodialysis treatment in 55 individuals with vascular disease and an age-matched group of controls (n = 21).

Pathologic mechanisms of type 1 VWD mutations R1205H and Y1584C through in vitro and in vivo mouse models.

Type 1 VWD is the mild to moderate reduction of VWF levels. This study examined the mechanisms underlying 2 common type 1 VWD mutations, the severe R1205H and more moderate Y1584C. In vitro biosynthesis was reduced for both mutations in human and mouse VWF, with the effect being more severe in R1205H.

A microRNA-regulated and GP64-pseudotyped lentiviral vector mediates stable expression of FVIII in a murine model of Hemophilia A.

The objective to use gene therapy to provide sustained, therapeutic levels of factor VIII (FVIII) for hemophilia A is compromised by the emergence of inhibitory antibodies that prevent FVIII from performing its essential function as a cofactor for factor IX (FIX). FVIII appears to be more immunogenic than FIX and an immune response is associated more frequently with FVIII than FIX gene therapy strategies. We have evaluated a modified lentiviral delivery strategy that facilitates liver-restricted transgene expression and prevents off-target expression in hematopoietic cells by incorporating microRNA (miRNA) target sequences.

Mutation-specific hemostatic variability in mice expressing common type 2B von Willebrand disease substitutions.

Type 2B von Willebrand disease (2B VWD) results from von Willebrand factor (VWF) A1 mutations that enhance VWF-GPIbalpha binding. These "gain of function" mutations lead to an increased affinity of the mutant VWF for platelets and the binding of mutant high-molecular-weight VWF multimers to platelets in vivo, resulting in an increase in clearance of both platelets and VWF. Three common 2B VWD mutations (R1306W, V1316M, and R1341Q) were independently introduced into the mouse Vwf cDNA sequence and the expression vectors delivered to 8- to 10-week-old C57Bl6 VWF(-/-) mice, using hydrodynamic injection.

A murine model for induction of long-term immunologic tolerance to factor VIII does not require persistent detectable levels of plasma factor VIII and involves contributions from Foxp3+ T regulatory cells.

Under certain instances, factor VIII (FVIII) stimulates an immune response, and the resulting neutralizing antibodies present a significant clinical challenge. Immunotherapies to re-establish or induce long-term tolerance would be beneficial, and an in-depth knowledge of mechanisms involved in tolerance induction is essential to develop immune-modulating strategies. We have developed a murine model system for studying mechanisms involved in induction of immunologic tolerance to FVIII in hemophilia A mice.

ADAMTS13 cleavage efficiency is altered by mutagenic and, to a lesser extent, polymorphic sequence changes in the A1 and A2 domains of von Willebrand factor.

The multimeric plasma protein von Willebrand factor (VWF) is regulated in size by its protease, ADAMTS13 (a disintegrin and metalloproteinase with thrombospondin type 1 motif, member 13). Y1605-M1606 cleavage site mutations and single nucleotide polymorphisms (SNPs) in the VWF A1 and A2 domains were examined for alteration in ADAMTS13-mediated cleavage of VWF. Recombinant human full-length VWF (rVWF) was digested with recombinant human ADAMTS13 (rADAMTS13) using a dialysis membrane method with 1.

Ex vivo gene therapy for hemophilia A that enhances safe delivery and sustained in vivo factor VIII expression from lentivirally engineered endothelial progenitors.

Novel therapeutic strategies for hemophilia must be at least as effective as current treatments and demonstrate long-term safety. To date, several small clinical trials of hemophilia gene transfer have failed to show the promise of preclinical evaluations. Therefore, we wanted to develop and evaluate the feasibility of a novel ex vivo gene transfer strategy whereby cells derived from progenitor cells are engineered to express factor VIII (FVIII) and then implanted subcutaneously to act as a depot for FVIII expression.

The mutational spectrum of type 1 von Willebrand disease: Results from a Canadian cohort study.

In order to evaluate the changes within the VWF gene that might contribute to the pathogenesis of type 1 von Willebrand disease (VWD), a large multicenter Canadian study was undertaken. We present data from the sequence analysis of the VWF gene in 123 type 1 VWD index cases and their families. We have identified putative mutations within the VWF gene in 63% (n = 78) of index cases, leaving 37% (n = 45) with no identified changes.

Cell type-specific regulation of von Willebrand factor expression by the E4BP4 transcriptional repressor.

Mechanisms of tissue-restricted patterns of von Willebrand factor (VWF) expression involve activators and repressors that limit expression to endothelial cells and megakaryocytes. The relative transcriptional activity of the proximal VWF promoter was assessed in VWF-producing and -nonproducing cells, and promoter activity was highest in endothelial cells followed by megakaryocytes. Only basal VWF promoter activity was seen in nonendothelial cells.

A novel type 2A von Willebrand factor mutation located at the last nucleotide of exon 26 (3538G>A) causes skipping of 2 nonadjacent exons.

In this manuscript, we describe a case of type 2A von Willebrand disease (VWD) caused by the novel heterozygous G>A transition at nucleotide 3538, which should result in the putative, nonconservative substitution of G1180R. This mutation was reproduced by site-directed mutagenesis; however, the recombinant mutant protein was efficiently secreted from cells and assembled correctly into multimers. Because the substitution is located at the last nucleotide of exon 26, the patient's platelet von Willebrand factor (VWF) mRNA was analyzed and 3 transcripts were observed: the normal transcript without the 3538G>A transition, a transcript with the in-frame deletion of exon 26, and a transcript with the in-frame deletions of exons 23 and 26.

Founder von Willebrand factor haplotype associated with type 1 von Willebrand disease.

To date, no dominant mutation has been identified in a significant proportion of patients with type 1 von Willebrand disease (VWD). In this study, we examined 70 families as part of the Canadian Type 1 VWD Study. The entire VWF gene was sequenced for 1 index case, revealing 2 sequence variations: intron 30 (5312-19A>C) and exon 28 at Tyr1584Cys (4751A>G).