Shawn Tinlin

Genetic linkage and association analysis in type 1 von Willebrand disease: results from the Canadian type 1 VWD study.

Summary.  Background: von Willebrand disease (VWD) is the most common bleeding disorder known in humans, with type 1 VWD representing the majority of cases. Unlike the other variant forms of VWD, type 1 disease represents a complex genetic trait, influenced by both genetic and environmental factors. Aim: To evaluate the contribution of the von Willebrand factor (VWF) and ABO blood group loci to the type 1 VWD phenotype, and to assess the potential for locus heterogeneity in this condition, we have performed genetic linkage and association studies on a large, unselected type 1 VWD population. Method: We initially collected samples from 194 Canadian type 1 VWD families for analysis. After the exclusion of families found to have either type 2 or type 3 VWD, and pedigrees with samples from single generations, linkage and association analysis was performed on 155 type 1 VWD families. Results and conclusion: The linkage study has shown a low heterogeneity LOD score of 2.13 with the proportion of families linked to the VWF gene estimated to be 0.41. Linkage was not detected to the ABO locus in this type 1 VWD population. In the family‐based association test, significant association was found between the type 1 VWD phenotype, the quantitative traits, VWF:Ag, VWF:RCo, and FVIII:C and the ABO ‘O’ and ‘A’ alleles and the VWF codon 1584 variant. There was also weak association with the −1185 promoter polymorphism and VWF:Ag, VWF:RCo, and FVIII:C plasma levels. These studies provide further evidence to support the role for genetic loci other than VWF and ABO in the pathogenesis of type 1 VWD.

In vivo evaluation of an adenoviral vector encoding canine factor VIII : High-level, sustained expression in hemophiliac mice

Hemophilia A is the most common severe hereditary coagulation disorder and is caused by a deficiency in blood clotting factor VIII (FVIII). Canine hemophilia A represents an excellent large animal model that closely mimicks the human disease. In previous studies, treatment of hemophiliac dogs with an adenoviral vector encoding human FVIII resulted in complete correction of the coagulation defect and high-level FVIII expression [Connelly et al. (1996). Blood 88, 3846]. However, FVIII expression was short term, limited by a strong antibody response directed against the human protein. Human FVIII is highly immunogenic in dogs, whereas the canine protein is significantly less immunogenic. Therefore, sustained phenotypic correction of canine hemophilia A may require the expression of the canine protein. In this work, we have isolated the canine FVIII cDNA and generated an adenoviral vector encoding canine FVIII. We demonstrate expression of canine FVIII in hemophiliac mice at levels 10-fold higher than those of the human protein expressed from an analogous vector. Canine FVIII expression was sustained above human therapeutic levels (50 mU/ml) for at least 1 year in hemophiliac mice.

Factors influencing therapeutic efficacy and the host immune response to helper‐dependent adenoviral gene therapy in hemophilia A mice

Summary.  Background: Adenoviral‐based methods of gene therapy have been ineffective at providing sustained factor (F)VIII expression in outbred populations of large animal hemophilic models primarily due to the immunogenicity of these vectors. Improvements have been made in vector design leading to the development of the helper‐dependent adenoviral (HD) system. Unfortunately, it remains unclear whether these modifications are sufficient to circumvent the induction of inhibitor formation associated with adenoviral gene transfer. Objective: To develop an HD vector capable of mediating sustained FVIII expression and to determine the variables that influence inhibitor development. Methods: HD vectors were constructed encoding the canine FVIII B‐domain deleted transgene under the control of either the cytomegalovirus (CMV) promoter or a tissue‐restricted hybrid element consisting of five HNF‐1 binding sites, located upstream of the human FVIII proximal promoter. Inbred and outbred populations of hemophilic mice were treated, and monitored for vector‐induced toxicity, therapeutic efficacy, and inhibitor formation. Results: When HD vectors utilizing the CMV promoter were administered, all hemophilic mice developed high levels of FVIII inhibitors. In contrast, vectors under the control of the HNF/FVIII element were capable of achieving sustained elevations of FVIII for over 6 months. Strain‐specific differences were also observed, with outbred animals showing a greater propensity towards inhibitor development in response to treatment. Conclusions: HD vectors can be used to provide long‐term FVIII expression in hemophilic animals, but treatment outcome and the induction of inhibitors is dependent on a number of variables including the transgene promoter, the vector dose, and the genetic background of the host.

The genetics of Canadian type 3 von Willebrand disease: further evidence for co-dominant inheritance of mutant alleles.

Type 3 von Willebrand disease (VWD) is the most severe form of the disease and is classically inherited in an autosomal recessive fashion.

Heterogeneity of the immune response to adenovirus‐mediated factor VIII gene therapy in different inbred hemophilic mouse strains

The development of anti‐factor VIII (FVIII) antibodies (inhibitors) is a critical concern when considering gene therapy as a potential treatment modality for hemophilia A. We used a hemophilia A mouse model bred on different genetic backgrounds to explore genetically controlled differences in the immune response to FVIII gene therapy.

Changes in the pattern of distribution of von Willebrand factor in rat aortic endothelial cells following thrombin generation in vivo

The pattern of distribution of von Willebrand factor (VWF) in relatively large sheets of rat aortic endothelial cells (EC) obtained by the Häutchen technique were analysed by immunocytochemistry and light microscopy. EC were examined pre and post administration of a procoagulant mixture of factor Xa (F.Xa) and phosphotidylcholine/phosphotidylserine (PCPS) vesicles which was demonstrated to result in the selective loss of high molecular weight multimers (HMWM) of plasma VWF in the rat. In placebo animals the pattern was heterogenous both in overall distribution and in individual cells which showed both a diffuse and granular pattern. Groups of intensely stained EC were oriented parallel to the longitudinal axis of the aorta and staining was particularly prominent around the orifices of the intercostal arteries, implicating shear‐stress as a possible factor in VWF expression by EC. Changes in the pattern of distribution of staining were observed at various time points post‐infusion of F.Xa/PCPS, suggesting the immediate release of VWF from EC stores followed by the recruitment of EC to synthesize and store VWF. These changes are consistent with the decrease in EC Weibel‐Palade body (WPB) content observed by EM in previously reported studies using this model.

A platelet release defect induced by aspirin or penicillin G does not increase gastrointestinal blood loss in thrombocytopenic rabbits

Summary. Gastrointestinal blood loss was compared in groups of normal and thrombocytopenic animals treated with medications known to induce qualitative platelet dysfunction. Thrombocytopenia was induced in rabbits by the intraperi‐toneal injection of busulphan dissolved in polyethylene glycol (PEG) at a dose of 60 mg/kg. Control animals received PEG alone; each group subsequently received daily intravenous injections of penicillin G, aspirin, sodium salicylate or isotonic saline. Mean daily gastrointestinal blood loss was determined by monitoring the appearance of 51Cr radioactivity in the faeces following the administration of 51Cr‐labelled erythrocytes prior to the administration of the test and control therapies. The administration of penicillin G was not associated with increased gastrointestinal blood loss in the thrombocytopenic animals as compared with the saline treated thrombocytopenic controls. Platelet aggregation studies confirmed the presence of a mild but significant defect in platelet aggregation. Aspirin produced a more pronounced defect in platelet aggregation but did not induce increased bleeding in the normal animals as compared with the controls, nor did it exacerbate the bleeding in thrombocytopenic animals. Sodium salicylate did not produce an aggregation defect and did not significantly modify gastrointestinal blood loss. It was concluded that drug‐induced qualitative platelet dysfunction does not necessarily increase bleeding through intact vessels despite previous evidence of a significant effect on platelet plug formation as monitored by the bleeding time.