Summary Background Targeting FVIII expression to platelets is a promising gene therapy approach for hemophilia A and is successful even in the presence of inhibitors. It is well known that platelets not only play important roles in hemostasis, but also in thrombosis and inflammation. Objective To evaluate whether platelet-FVIII expression might increase the risk for thrombosis and thereby compromise the safety of this approach. Methods In this study, platelet-FVIII expressing transgenic mice were examined either in steady state or under prothrombotic conditions induced by inflammation or the factor V Leiden mutation. Native whole blood thrombin generation assay, ROTEM analysis, and ferric chloride induced vessel injury were used to evaluate the hemostatic properties. Various parameters associated with the thrombosis risk, including D-Dimer, thrombin anti-thrombin complexes, fibrinogen, tissue fibrin deposition, platelet activation status and activatability, and platelet-leukocyte aggregates, were assessed. Results We generated a new line of transgenic mice that expressed 30-fold higher platelet-FVIII levels than therapeutically required to restore hemostasis in hemophilic mice. In steady state as well as under prothrombotic conditions induced by LPS-mediated inflammation or the factor V Leiden mutation, supratherapeutic levels of platelet-FVIII did not appear thrombogenic. Furthermore, FVIII-expressing platelets were neither hyper-activated nor hyper-activatable upon agonist activation. Conclusion We conclude that in mice, more than 30-fold higher platelet FVIII levels than required for therapeutic efficacy in hemophilia A are not associated with a thrombotic predilection.
Plant molecular phylogeneticists have supported an analytical approach of combining loci from different genomes, but the combination of mitochondrial sequences with chloroplast and nuclear sequences is potentially problematic. Low substitution rates in mitochondrial genes should decrease saturation, which is especially useful for the study of deep divergences. However, individual mitochondrial loci are insufficiently informative, so that combining congruent loci is necessary. For this study atp1 and cox1 were selected, which are of similar lengths, encode components of the respiratory pathway, and generally lack introns. Thus, these genes might be expected to have similar functional constraints, selection pressures, and evolutionary histories. Strictly parallel sampling of 52 species was achieved as well as six additional composite terminals with representatives from the major angiosperm clades. However, analyses of the separate loci produced strongly incongruent topologies. The source of the incongruence was investigated by validating sequences with questionable affinities, excluding RNA-edited nucleotides, deleting taxa with unexpected phylogenetic associations, and comparing different phylogenetic methods. However, even after potential artifacts were addressed and sites and taxa putatively associated with conflict were excluded, the resulting gene trees for the two mitochondrial loci were still substantially incongruent by all measures examined. Therefore, combining these loci in phylogenetic analysis may be counterproductive to the goal of fully resolving the angiosperm phylogeny.
Key Points A novel HA rat model caused by an inversion exhibits a severe spontaneous bleeding phenotype. The severe spontaneous bleeding phenotype in HA rats is rescued by platelet-targeted FVIII expression.
Gene therapy may lead to a cure for hemophilia B (HB) if it is successful. Data from clinical trials using adeno-associated virus (AAV)–mediated liver-targeted FIX gene therapy are very encouraging. However, this protocol can be applied only to adults who do not have liver disease or anti-AAV antibodies, which occur in 30% to 50% of individuals. Thus, developing a protocol that can be applied to all HB patients is desired. Our previous studies have demonstrated that lentivirus-mediated platelet-specific FIX (2bF9) gene therapy can rescue bleeding diathesis and induce immune tolerance in FIXnull mice, but FIX expression was only ∼2% to 3% in whole blood. To improve the efficacy, we used a codon-optimized hyperfunctional FIX-Padua (2bCoF9R338L) to replace the 2bF9 cassette, resulting in 70% to 122% (35.08-60.77 mU/108 platelets) activity levels in 2bCoF9R338L-transduced FIXnull mice. Importantly, sustained hyperfunctional platelet-FIX expression was achieved in all 2bCoF9R338L-transduced highly immunized recipients with activity levels of 18.00 ± 9.11 and 9.36 ± 12.23 mU/108 platelets in the groups treated with 11 Gy and 6.6 Gy, respectively. The anti-FIX antibody titers declined with time, and immune tolerance was established after 2bCoF9R338L gene therapy. We found that incorporating the proteasome inhibitor bortezomib into preconditioning can help eliminate anti-FIX antibodies. The bleeding phenotype in 2bCoF9R338L-transduced recipients was completely rescued in a tail bleeding test and a needle-induced knee joint injury model once inhibitors dropped to undetectable. The hemostatic efficacy in 2bCoF9R338L-transduced recipients was further confirmed by ROTEM and thrombin generation assay (TGA). Together, our studies suggest that 2bCoF9R338L gene therapy can be a promising protocol for all HB patients, including patients with inhibitors.
Type 2N von Willebrand disease is caused by mutations in the factor VIII (FVIII) binding site of von Willibrand factor (VWF), resulting in dysfunctional VWF with defective binding capacity for FVIII. Here we developed a novel type 2N mouse model using CRISPR/Cas9 technology. In homozygous VWF2N/2N mice, plasma VWF levels were normal (1167±257 mU/ml) but the VWF was completely incapable of binding FVIII, resulting in 53±23 mU/ml of plasma FVIII levels that were similar to those in VWF deficient (VWF-/-) mice. When wild-type human or mouse VWF was infused into VWF2N/2N mice, endogenous plasma FVIII was restored, peaking at 4-6 hours post-infusion, demonstrating that FVIII expressed in VWF2N mice is viable, but short-lived unprotected in plasma due to dysfunctional 2N-VWF. The whole blood clotting time and thrombin generation were impaired in VWF2N/2N but not in VWF-/- mice. The bleeding time and blood loss in VWF2N/2N mice were similar to wild-type mice in the lateral tail vein or ventral artery injury model. However, VWF2N/2N, but not VWF-/- mice, lost a significant amount of blood during the primary bleeding phase after a tail tip amputation injury model, indicating that there are other alternative pathway(s) that can at least partially restore hemostasis when VWF is absent. In summary, we have developed a novel mouse model by gene editing with both the pathophysiology and clinical phenotype found in severe type 2N patients. This unique model can be used to investigate the biological properties of VWF/FVIII association in hemostasis and beyond.
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