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Arthur R Thompson - One of the best experts on this subject based on the ideXlab platform.
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naked dna transfer of Factor viii induced transgene specific species independent immune response in hemophilia a mice
Molecular Therapy, 2004Co-Authors: David Lillicrap, Arthur R Thompson, Rita Sarkar, Zhenping Shen, Randal J Kaufman, Hans D Ochs, David J Rawlings, Carol H MiaoAbstract:The development of antibodies to a previously unexpressed protein product may limit the success of human gene therapy approaches. We inserted B-domain-deleted Factor VIII (FVIII) cDNA of human, canine, or murine origin into the multiple cloning site of a liver-specific vector, pBS-HCRHPI-A, to yield plasmids pBS-HCRHPI-FVIIIA, pBS-HCRHPI-cFVIIIA, and pBS-HCRHPI-mFVIIIA, respectively. Fifty micrograms of each plasmid in 2 ml of solution was rapidly injected into the tail vein of three groups of hemophilia A mice. Factor VIII levels ranging from 3 to 12 IU/ml were obtained from all three groups (normal is 1 IU/ml in human plasma) 3 days after treatment. These initial very high levels of functional human, canine, or murine Factor VIII, however, fell gradually to undetectable levels within 2–3 weeks, and their disappearance correlated with the generation of high-titer, inhibitory anti-FVIII antibodies. Notably, this immune response occurred independent of the species of origin of the Exogenous Factor VIII. Antibody titers to Factor VIII were detected beginning at 2 weeks, reached a plateau and remained at high levels for over 6 months. The majority of anti-hFVIII IgG was IgG1 isotype specific, suggesting a humoral response mediated by Th2-induced signals. Consistent with this idea, in a separate group of mice treated with pBS-HCRHPI-FVIIIA, transient immunosuppression by cyclophosphamide significantly delayed (5/6) or abolished (1/6) inhibitory antibody formation against the transgene.
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loss of tolerance to Exogenous and endogenous Factor viii in a mild hemophilia a patient with an arg593 to cys mutation
Blood, 1997Co-Authors: Arthur R Thompson, Michael E P Murphy, Miaoliang Liu, Evgueni L SaenkoAbstract:A 42-year-old patient with mild hemophilia A developed spontaneous muscle hematomas 1 month after intense therapy with Factor VIII concentrates. Factor VIII clotting activity was less than 1% and his Factor VIII inhibitor was 10 Bethesda units (BU)/mL. The titer peaked at 128 BU despite daily infusions of Factor VIII; 1 year later, the titer was 13 BU with no spontaneous bleeding for 4 months. The plasma inhibitor was 95% neutralized by Factor VIII A2 domain but less than 15% neutralized by light-chain or C2 domain. His inhibitor did not cross-react with porcine Factor VIII and was at least 10-fold less reactive to a series of hybrid Factor VIII proteins in which human residues 484-508 are replaced by the homologous porcine sequence (Healey et al, J Biol Chem 270:14505, 1995). The inhibitor patient's DNA encoding his A2 domain and flanking sequences showed a C-T transition predicting Arg593 to Cys. Thirteen patients from 5 unrelated families with Cys593 have not developed inhibitors. Factor VIII clotting activity from one of them was inhibited similarly to diluted normal plasma by inhibitor patient plasma. In an homologous structure, ceruloplasmin (Zaitseva et al, J Biol Inorgan Chem 1:15,1996), the residue equivalent to Arg593, is in a loop distinct from residues 484-508. On solution phase immunoprecipitation with labeled Factor VIII fragments, A2, light chain, and C2 domains bound. In contrast to typical immune responses to Factor VIII in patients with severe hemophilia A, this patient's inhibitor was almost entirely reactive with common epitopes within the A2 domain whereas by more sensitive immunoprecipitation testing antibodies to light chain epitopes were also present. Accordingly, immune responsiveness to Exogenous Factor VIII (antigen burden) appears to be more critical than his endogenous, hemophilic Factor VIII to his developing high-titer anti–Factor VIII antibodies and loss of tolerance to both native and hemophilic Factor VIII proteins.
Margareth C Ozelo - One of the best experts on this subject based on the ideXlab platform.
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heat treatment of samples improve the performance of the nijmegen bethesda assay in hemophilia a patients undergoing immune tolerance induction
Thrombosis Research, 2015Co-Authors: Silmara Aparecida De Lima Montalvao, Alini Camargo Tucunduva, Andrea Luisa De Almeida Sambo, Erich Vinicius De Paula, Samuel De Souza Medina, Margareth C OzeloAbstract:Abstract Nijmegen–Bethesda assay is the gold standard to assess inhibitory antibodies against Factor (F) VIII. This method has some limitations, including high coefficient of variation and possible interference of residual endogenous or Exogenous Factor VIII. Heat-treatment of samples at 56 °C for 30 min could be a strategy to improve the sensitivity of this test. The aim of this study was to compare inhibitor quantification in hemophilia patients with and without inhibitor performed in previously heated and non-heated samples. A total of 109 analyses from 46 patients with severe hemophilia A were performed. Patients were divided into three groups: 20 patients with no history of inhibitor, recently and not recently exposed to FVIII (group I), 21 patients with history of inhibitor not exposed to FVIII (group II), and 5 patients (68 samples) undergoing an immune tolerance induction (ITI) protocol (group III). For patients with no history of inhibitor, heat-treatment did not modify the results (p = 0.24). However, differences in inhibitor levels between heated and non-heated samples were observed in patients with history of inhibitor (group II, p
Erin L Kuether - One of the best experts on this subject based on the ideXlab platform.
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lentivirus mediated platelet gene therapy of murine hemophilia a with pre existing anti Factor viii immunity
Journal of Thrombosis and Haemostasis, 2012Co-Authors: Erin L Kuether, Jocelyn A Schroeder, Scot A Fahs, Brian C Cooley, Yingyu Chen, Robert R Montgomery, David A WilcoxAbstract:Summary. Background: The development of inhibitory antibodies, referred to as inhibitors, against Exogenous Factor VIII in a significant subset of patients with hemophilia A remains a persistent challenge to the efficacy of protein replacement therapy. Our previous studies using the transgenic approach provided proof-of-principle that platelet-specific expression could be successful in treating hemophilia A in the presence of inhibitory antibodies. Objective: To investigate a clinically translatable approach for platelet gene therapy of hemophilia A with pre-existing inhibitors. Methods: Platelet FVIII expression in preimmunized FVIIInull mice was introduced by transplantation of lentivirus-transduced bone marrow or enriched hematopoietic stem cells. FVIII expression was determined with a chromogenic assay. The transgene copy number per cell was quantitated with real-time PCR. Inhibitor titer was measured with the Bethesda assay. Phenotypic correction was assessed by the tail clipping assay and an electrolytically induced venous injury model. Integration sites were analyzed with linear amplification-mediated PCR. Results: Therapeutic levels of platelet FVIII expression were sustained in the long term without evoking an anti-FVIII memory response in the transduced preimmunized recipients. The tail clip survival test and the electrolytic injury model confirmed that hemostasis was improved in the treated animals. Sequential bone marrow transplants showed sustained platelet FVIII expression resulting in phenotypic correction in preimmunized secondary and tertiary recipients. Conclusions: Lentivirus-mediated platelet-specific gene transfer improves hemostasis in mice with hemophilia A with pre-existing inhibitors, indicating that this approach may be a promising strategy for gene therapy of hemophilia A even in the high-risk setting of pre-existing inhibitory antibodies.
Holm Schneider - One of the best experts on this subject based on the ideXlab platform.
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recurrent inversion with concomitant deletion and insertion events in the coagulation Factor viii gene suggests a new mechanism for x chromosomal rearrangements causing hemophilia a
Human Mutation, 2007Co-Authors: Christiane Muhle, Martin Zenker, Nadia Chuzhanova, Holm SchneiderAbstract:Recurrent int22h-related inversions in the coagulation Factor VIII gene (F8) are the most common cause of severe hemophilia A. Such inversions have repeatedly been hypothesized to be associated with concomitant deletions that are responsible for an increased risk of immune responses against therapeutic Exogenous Factor VIII. However, exact DNA breakpoints have not yet been reported. In a patient with persistent Factor VIII-inactivating antibodies, molecular analysis of F8 including Southern Blot, long-range PCR and primer walking techniques revealed a combination of an int22h2-related inversion, deletion of exons 16-22 and insertion of a duplicated part of the X-chromosomal MPP1 gene. This novel genomic rearrangement was also detectable in the patient's mother, but absent in both maternal grandparents. The genetic defect most likely originated from a complex X-chromosomal recombination event during spermatogenesis due to the formation of a DNA loop stabilized by Alu and LINE repeat elements. Elucidation of such combined mutations may allow early identification of patients at high risk of developing Factor VIII-neutralizing antibodies and will help to understand the mechanisms behind gross chromosomal rearrangements causing hemophilia A and other diseases.
John K Pasi - One of the best experts on this subject based on the ideXlab platform.
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aav5 Factor viii gene transfer in severe hemophilia a
The New England Journal of Medicine, 2017Co-Authors: Savita Rangarajan, Liron Walsh, Will Lester, David J Perry, Bella Madan, Michael Laffan, Christian Vettermann, Glenn F Pierce, Wing Yen Wong, John K PasiAbstract:BackgroundPatients with hemophilia A rely on Exogenous Factor VIII to prevent bleeding in joints, soft tissue, and the central nervous system. Although successful gene transfer has been reported in patients with hemophilia B, the large size of the Factor VIII coding region has precluded improved outcomes with gene therapy in patients with hemophilia A. MethodsWe infused a single intravenous dose of a codon-optimized adeno-associated virus serotype 5 (AAV5) vector encoding a B-domain–deleted human Factor VIII (AAV5-hFVIII-SQ) in nine men with severe hemophilia A. Participants were enrolled sequentially into one of three dose cohorts (low dose [one participant], intermediate dose [one participant], and high dose [seven participants]) and were followed through 52 weeks. ResultsFactor VIII activity levels remained at 3 IU or less per deciliter in the recipients of the low or intermediate dose. In the high-dose cohort, the Factor VIII activity level was more than 5 IU per deciliter between weeks 2 and 9 after ...