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CHS Research Grants for 2013

Incorporation of rFVIII into platelets as a potential therapy in patients with inhibitory antibodies to factor VIII

Dr. Walter Kahr
The Hospital for Sick Children – Toronto, Ontario
First year funding

Current treatment of severe hemophilia A patients in Canada involves regular injections of recombinant factor VIII (rFVIII). This treatment is effective, but rFVIII injections are usually frequent because factor does not stay long in the blood stream and around 25% of patients develop inhibitors that block rFVIII effectiveness. Work is underway to develop longer-lasting forms of rFVIII and limit the effects of inhibitors. One approach involves platelets, the tiny, abundant blood cells that coordinate blood clotting. Platelets normally do not contain FVIII, but genetic engineering has been used to make mouse platelets containing FVIII that have proven to be effective in preventing bleeding in hemophilic mice even when they have inhibitors. We have been working on ways to load platelets with rFVIII that do not involve the risk, expense and uncertainties of genetic engineering. Our preliminary work shows that this is possible, and that rFVIII-loaded platelets can inhibit bleeding in hemophilic mice. The goal of our proposed project is to improve our methods for making rFVIII-loaded platelets and to demonstrate their potential for use in hemophilia patients with inhibitors, who are often difficult to treat with conventional therapies.

Novel imaging techniques for assessment of early cartilage and soft tissue changes in haemophilic ankles

Dr. Andrea Doria
The Hospital for Sick Children – Toronto, Ontario
Second year funding continued in 2013 due to late start.

Co-investigators: Dr. Aaron Fenster; Dr. Marshall Sussman; Dr. Victor Blanchette

Hemophilia is an inherited bleeding disorder characterized by the lack of coagulation factors which results in an inability to control bleeding into joints, leading to long-term joint damage. Prophylaxis, reduces the joint symptoms and avoids further degeneration of the joints, however it should be started prior to the development of cartilage lesions. Repeated extravazation of blood into the joint cavity is the factor responsible for cartilage degeneration in hemophilic arthropathy. Microstructural cartilage changes are thought to precede macroscopic cartilage lesions [8] which are responsible for most of the morbidity of hemophilic arthropathy. Conventional imaging techniques are unable to visualize early soft tissue and cartilage changes. Evaluation of soft tissue changes and microstructural cartilage changes with sensitive imaging tools may direct clinical management and prophylaxis towards avoiding further irreversible macroscopic osteochondral damage. New functional MRI techniques and 3DUS anatomic imaging may be able to diagnose early joint changes at a time where treatment is still effective to avoid further degeneration of the joint. No prior studies have investigated the imaging of very early structural and physiologic events in hemophilic joints. We will pioneer the development of novel imaging techniques for assessment of early soft tissue and cartilage changes in hemophiliacs.

Oral administration of FVIII DNA to modulate inhibitors in hemophilia mice

Dr. Gonzalo Hortelano
McMaster University – Hamilton, Ontario
Second year funding

The delivery of genes as therapeutic drugs is still facing numerous challenges, particularly how to effectively deliver a therapeutic gene to the desired target cells, and the immune responses generated by the immune system to foreign DNA and to foreign or new proteins. Viruses are the most popular vehicle to deliver therapeutic genes, since they are very efficient at infecting cells. However, some human trials have revealed some safety concerns that need to be addressed. Additional and novel ways to deliver therapeutic genes would be desirable.
Ingestion of therapeutic genes would allow the intestine to produce a therapeutic protein for distribution throughout the body. However, DNA is degraded in the acidic stomach environment and by DNA-degrading proteins in the intestine. Our laboratory has shown that DNA formulated with chitosan into nanoparticles can withstand degradation under those conditions. Interestingly, we have not observed antibodies against the therapeutic protein encoded by ingested DNA. Further, we have evidence suggesting that this strategy can modulate the immune response to FVIII. The overall goal of this proposal is to understand the effect of ingested DNA on the immune system and to evaluate the potential of oral DNA in preventing and/or reducing FVIII inhibitors.

Platelet–type von Willebrand disease: Novel studies in the PT-VWD mouse model

Dr. Maha Othman
Queen’s University – Kingston, Ontario
Second year funding.

PT-VWD is a rare inherited bleeding disorder .Only 55 patients are described worldwide. The disease results from a hyper-responsive platelet protein known as GP Ib alpha due to genetic defect in the platelet GP1BA gene. The disease is characterized by low plasma VWF and platelet count. The latter can be aggravated in response to stress, infection and during pregnancy. Patients can have life threatening bleeding if not diagnosed and treated properly.
Currently the standard test for evaluating bleeding pattern is VWF:RCo (ristocetin cofactor assay) which has shown problems in relation to sensitivity and ristocetin binding. We hypothesize that the thromboelastography (TEG); a global hemostatic test is useful in evaluating hemostasis in PT-VWD and would potentially help monitor patients during pregnancy, at times of infection and following treatment. We plan to carry out novel studies in the PT-VWD
mouse model carrying a common human mutation G233V and exhibiting a similar phenotype to the human disease. We will investigate the utility of TEG in PT-VWD, evaluate the mouse model during early and late pregnancy and in response to infection and evaluate the effect of GP1b alpha inhibitors in improving the bleeding phenotype in these mice. These studies have not been previously done and are likely to improve our understanding of the PT-VWD disease and help diagnosis and follow up of patients.

The effect of FVIII on complement system activation

Lab work studentship

Joshua Foley
University of British Columbia
Under the supervision of Dr. Edward Conway
Summer 2013 funding

The blood clotting system is activated upon blood vessel damage and as a result blood loss is minimized. Individuals with hemophilia A lack an essential blood clotting factor and as a result they are at risk of excessive bleeding. State-of-the-art hemophilia treatment involves replenishing the missing clotting factor called factor VIII. Unfortunately, some of the patients receiving replacement FVIII develop antibodies, which neutralizes the treatment. This is the biggest complication of hemophilia treatment in developed countries and patients with these neutralizing antibodies require very expensive therapies. Recently, many novel connections between the blood clotting system and the complement system have been identified. The complement system is the first line of defense against invading pathogens and its activation has been shown to increase the body’s immune response. The purpose of this project is to determine the effect of FVIII on complement activation with the hope of identifying mechanisms that increase the immune response. The data generated during this project will guide future studies aimed at unraveling mechanisms contributing to neutralizing antibody development in hemophilia.

In-vitro clotting assays measuring equivalent concentration of FEIBA and rFVIIa

Lab work studentship

Xena Li
McMaster University
Under the supervision of Dr. Howard Chan, McMaster University – Hamilton, Ontario
Summer 2013 funding

Hemophilia is an inherited bleeding disorder due to the deficiency of a clotting protein. Although the usual treatment is to replace the clotting protein, some patients may become refractory to the therapy because it is rejected by patients' own immune system. FEIBA and rFVIIa are two treatment options, but the equivalent dosing comparing these two products is not yet available. Without this information, it is not possible to evaluate which product has better effects. Our project will use some assays measuring global clot formation to determine the concentrations at which these products show equivalent clotting ability.

Investigating the Influence of SNPs (Single Nucleotide Polymorphisms) on variability of the Type 2B VWD phenotype

Lab work studentship

Malak Elbatarny
Queen’s University
Under the supervision of Dr. Paula James, Queen’s University – Kingston, Ontario
Summer 2013 funding

Type 2B von Willebrand disease (VWD) is an inherited bleeding disorder caused by mutations in the VWF gene that alter the function of a protein that is critical to clotting (von Willebrand factor). As a result, individuals who have the disease have moderate bleeding symptoms in mucocutaneous tissue, low platelet counts and low levels of the affected protein, von Willebrand factor (VWF). Interestingly, there is substantial variability among the symptoms of these patients that are not fully understood. In this study, we will attempt to investigate correlations between genotype and phenotype (the severity of the disease) using what is known as Family-Based Association Testing (FBAT). We will identify Single Nucleotide Polymorphisms (SNPs) or single base changes occurring in three large families with Type 2B VWD (27 individuals in total), and look for relationships between presence of these identified SNPs and the family members’ phenotypes. We expect to find that certain SNPs may be correlated with the severity of the patients’ disease including the bleeding symptoms, VWF levels and/or platelet count.