A novel mouse model of type 2N VWD was developed by CRISPR/Cas9 gene editing and recapitulates human type 2N VWD.

Type 2N von Willebrand disease is caused by mutations in the factor VIII (FVIII) binding site of von Willebrand factor (VWF), resulting in dysfunctional VWF with defective binding capacity for FVIII. 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 to 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. 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 mice, but not VWF-/- mice, lost a significant amount of blood during the primary bleeding phase after a tail tip amputation injury model, indicating that alternative pathways 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.

Platelet-targeted hyperfunctional FIX gene therapy for hemophilia B mice even with preexisting anti-FIX immunity.

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.

The severe spontaneous bleeding phenotype in a novel hemophilia A rat model is rescued by platelet FVIII expression.

Previous studies have shown that platelet-specific factor VIII (FVIII) expression (2bF8) restores hemostasis and induces immune tolerance in hemophilia A (HA) mice even with preexisting inhibitors. Here we investigated for the first time whether platelet FVIII expression can prevent severe spontaneous bleeding in rat HA, a model mimicking the frequent spontaneous bleeding in patients with severe HA. A novel FVIII-/- rat model in a Dahl inbred background (Dahl-FVIII-/-) with nearly the entire rat FVIII gene inverted was created by using a CRISPR/Cas9 strategy. There was no detectable FVIII in plasma. Spontaneous bleeding in the soft tissue, muscles, or joints occurred in 100% of FVIII-/- rats. Sixty-one percent developed anti-FVIII inhibitors after ≥2 doses of recombinant human FVIII infusion. However, when 2bF8 transgene was crossed into the FVIII-/- background, none of the resulting 2bF8tg+FVIII-/- rats (with platelet FVIII levels of 28.26 ± 7.69 mU/108 platelets and undetectable plasma FVIII) ever had spontaneous bleeding. When 2bF8tg bone marrow (BM) was transplanted into FVIII-/- rats, only 1 of 7 recipients had a bruise at the early stage of BM reconstitution, but no other spontaneous bleeding was observed during the study period. To confirm that the bleeding diathesis in FVIII-/- rats was ameliorated after platelet FVIII expression, rotational thromboelastometry and whole-blood thrombin generation assay were performed. All parameters in 2bF8tg BM transplantation recipients were significantly improved compared with FVIII-/- control rats. Of note, neither detectable levels of plasma FVIII nor anti-FVIII inhibitors were detected in 2bF8tg BM transplantation recipients. Thus, platelet-specific FVIII expression can efficiently prevent severe spontaneous bleeding in FVIII-/- rats with no anti-FVIII antibody development.

A rat model of severe VWD by elimination of the VWF gene using CRISPR/Cas9.

BACKGROUND: Von Willebrand Disease (VWD) is the most common inherited bleeding disorder, caused by quantitative and qualitative changes in von Willebrand factor (VWF). The biology of VWD, studied in canine, porcine, and murine models, differ in species-specific biology of VWF and the amenability to experimental manipulations such as phlebotomy. The factor VIII (FVIII) levels in these models are higher than in humans with type 3 VWD, suggesting functional differences between FVIII and VWF.ObjectivesTo develop a VWF knock out (VWF-/-) rat by excision of all 52 exons of the VWF locus. METHODS: The entire VWF gene was eliminated in Sprague-Dawley (Crl:SD) rats via CRISPR/Cas9-mediated gene editing. VWF antigen (VWF:Ag), VWF propeptide, and VWF collagen IV binding (VWF:CB4) levels were determined by ELISA assays and FVIII chromogenic activity (FVIII:C) levels by chromogenic FVIII assays. Lateral tail veins were transected to measure bleeding time. VWF-/- rats were infused with FVIII-/- rat platelet poor plasma (PPP) to determine response of plasma FVIII. RESULTS: Breeding of VWF ± rats yielded VWF-/- offspring at normal Mendelian ratios. VWF:Ag, VWF propeptide, VWF:CB4, and FVIII:C plasma levels were undetectable in VWF-/- rats. VWF-/- rats bled longer and more than VWF+/- and VWF+/+ rats when challenged. Transfusion of FVIII-deficient platelet-poor plasma induced a rapid rise in endogenous FVIII:C in VWF-/- rats. CONCLUSION: This rat model of severe VWD due to elimination of the entire VWF gene recapitulates the severe secondary deficiency of FVIII seen in human type 3 VWD and facilitates the study of VWF and FVIII and their interactions.

Phylogenetic analyses of two mitochondrial metabolic genes sampled in parallel from angiosperms find fundamental interlocus incongruence.

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.