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

2022 ◽  
Author(s):  
Qizhen Shi ◽  
Scot A Fahs ◽  
Jeremy G Mattson ◽  
Hongyin Yu ◽  
Crystal L Perry ◽  
...  
Keyword(s):  
Mouse Model ◽  
Gene Editing ◽  
Binding Capacity ◽  
Alternative Pathway ◽  
Biological Properties ◽  
Von Willebrand Disease ◽  
Wild Type ◽  
Injury Model ◽  
Post Infusion ◽  
Tail Tip

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.

Recombinant Protein Infusion and Hydrodynamic Plasmid Delivery Strategies Provide Distinct and Complementary Approaches to the Characterization of Variant Forms of Von Willeband Factor (VWF) in VWF Knockout Mice.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 2020-2020
Author(s):  
Cynthia M. Pruss ◽  
Carol A. Hegadorn ◽  
Andrea Labelle ◽  
Erin Burnett ◽  
Mia Golder ◽  
...  
Keyword(s):  
Molecular Weight ◽  
Recombinant Protein ◽  
High Molecular Weight ◽  
Significant Proportion ◽  
Von Willebrand Disease ◽  
Hek293 Cells ◽  
Wild Type ◽  
Platelet Counts ◽  
Post Infusion ◽  
Von Willebrand

Abstract Von Willebrand Factor (VWF) is a large multimeric glycoprotein that mediates platelet adhesion to the damaged blood vessel wall and subsequent platelet aggregation at the site of vascular injury. The size of VWF multimers is regulated by the metalloprotease, ADAMTS13. Alterations in VWF sequence can lead to an increase or decrease in ADAMTS13-mediated cleavage, resulting in either a loss or increase in high molecular weight VWF multimers, respectively. With the availability of a VWF knockout mouse, variant forms of VWF can be evaluated in vivo in terms of their contribution to hemostasis and thrombosis. In these studies, we have taken into account the significant differences in VWF-GPIb binding and ADAMTS13 cleavage efficiency seen between mice and humans and have also assumed that functionally important residues are likely to be conserved between species. With these considerations in mind, the protocols described in this report utilize mouse-exclusive reagents. Previous reports of correction of the VWF KO phenotype using hydrodynamic gene delivery have shown contradictory results for correction of the bleeding time and blood volume loss as well as a grossly abnormal multimer structure of the rescued VWF protein, due initially to an inadvertent C799R mutation and latterly to factors possibly related to the site of VWF synthesis. In this study, we compare wild type VWF clearance to a cleavage site knockout, Y1605A/M1606A, a type 2A Von Willebrand Disease (VWD) mutation, R1597W, and the common type 1 VWD mutation, Y1584C. The murine R1597W variant exhibited increased ADAMTS13-mediated cleavage (0.36- fold ADAMTS13 concentration), and the Y1605A/M1606A variant greatly decreased cleavage in vitro (>100-fold ADAMTS13 concentration). VWF KO mice, 7-10 weeks old, were injected with recombinant murine VWF (200U/kg) produced in HEK293 cells. VWF antigen levels (VWF:Ag), multimers, and complete blood counts (CBCs) were performed. Compared to the wild type infused protein (T1/2=33.2 minutes), the Y1605A/M1606A and R1597W mutant proteins show faster clearance (T1/2=17.7 minutes, p<0.001, and 27.5 minutes, p=0.025, respectively). Although, surprisingly, the Y1605A/M1606A variant showed a preferential loss of high molecular weight material compared to wild type recombinant protein, the R1597W type 2A mutant had a much more rapid loss of the high molecular multimers compared to the other proteins analyzed. No statistical differences were observed for platelet counts or other CBC parameters post protein infusion regardless of mutation compared to resting VWF KO mice. Hydrodynamic injections of a plasmid containing the ubiquitous synthetic CAG promoter and wild type VWF were performed on eight week old mice, with delivery of 100μg plasmid DNA in 10% body weight Ringer’s solution over 5-7 seconds. Maximum VWF:Ag levels of 10.67 U/ml were observed two days post infusion, with a significant proportion of observable high molecular weight VWF, indistinguishable from normal C57BL6 mouse plasma pool. Mouse FVIII:C levels follow a similar trend over the time course with maximum levels observed on day 3 at 206.7% activity. Mouse platelet counts were affected, with lower platelet counts rebounding to normal levels by day 7. No other changes in CBCs were observed. Intriguingly, none of the recombinant forms of VWF nor the hydrodynamically produced VWF protein show the typical triplet structure observed in normal mouse and human plasma, regardless of circulation time. The recombinant proteins migrate with the central band of the multimer triplet, while that of the hydrodynamic protein migrates with the lower triplet band, showing a lower molecular weight. These differences could be due to changes in protein structure or glycosylation from normally produced platelet and endothelial VWF. CAG-mVWF DNA Hydrodynamic Injection Results Days post infusion VWF:Ag (U/ml) FVIII:C (%) Platelets (103/μl) All data presented as value ± SD from 2-3 mice. Resting values are from at least 20 VWF KO mice. 1 4.82±0.18 62±16.7 440±101 2 10.67±1.06 128.5±53.6 547±30 3 9.92±0.53 206.7±29.4 558±39 7 1.03±0.47 78.3±3.3 699±53 10 0.51±0.53 30.4±16 502±189 14 0.13±0.08 26.8±0 470±276 Resting 0.0±0.0 (-) 569±167

scholarly journals Defects in Sialic Acid Biosynthesis Cause Dysregulation of the Alternative Pathway of Complement

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 3-3
Author(s):  
Hang Chen ◽  
Luyi Peng ◽  
Jia Yu ◽  
Xuan Yuan ◽  
Shruti Chaturvedi ◽  
...  
Keyword(s):  
Sialic Acid ◽  
Cell Surface ◽  
De Novo ◽  
Binding Capacity ◽  
Alternative Pathway ◽  
Targeted Sequencing ◽  
Wild Type ◽  
Advisory Committees ◽  
De Novo Biosynthesis ◽  
Alternative Pathway Of Complement

Introduction: Atypical Hemolytic Uremic Syndrome (aHUS) is a disease characterized by microangiopathic hemolytic anemia, thrombocytopenia, and acute kidney injury. aHUS is usually caused by a predisposing germline variant in a complement regulatory gene, and a second hit of a complement amplifying condition such as infection, pregnancy, or inflammation.However, only approximately 50% of aHUS patients have identifiable genetic variants, with variants in factor H (CFH) accounting for 20%-30% of the genetic predisposition, C3 for 7%, membrane cofactor protein (MCP)/CD46 for 8%, factor B (CFB) for 2% and factor I (CFI) for 6%. For the other 50% of the patients, the genetic predisposition remains elusive. CFH binds to α2,3 sialic acid (SA) linked glycans on host cell surfaces and protects against attack by the alternative pathway of complement (APC). We hypothesized that the biosynthesis of SA is essential to complement regulation and SA biosynthesis defects predispose to aHUS. Methods: We performed targeted sequencing on 34 aHUS patients and 43 healthy controls for 4 genes that are responsible for the de novo biosynthesis of sialic acid: GNE, NANS, NANP, and CMAS. Then we used CRISPR-Cas9 and lentivirus systems to manipulate these genes in TF1 or TF1 PIGA-null cells, which lack the glycophosphatidylinositol-linked cell surface complement regulators CD55 and CD59, and allow the APC cascade to proceed once activated. α2,3 SA levels on the cell surface were measured with Maackia Amurensis lectin II (MAL II). Finally, we studied the functional consequences of these genetic changes. Normal human serum (NHS) was used to activate the APC on cells, and factor D (CFD) depleted serum (D-Dpl) was used to specifically block APC activation. C5b-9 deposition and CFH binding capacity on cells were detected by flow cytometry, and complement induced cell killing was detected via a WST-1 cell viability assay (mHam). Results: i) Rare germline variants found in SA biosynthesis genes Two rare germline variants (minor allele frequency < 0.005; data from GnomAD) were identified via targeted sequencing. An NANS M117I variant was found in an aHUS patient, while an NANP A153V variant was found in a control. The aHUS case did not harbor any variants in known complement genes. ii) Loss ofNANS but not NANP decreasesα2,3 SA on TF1 cells NANS knockout TF1 cells showed decreased α2,3 SA, demonstrating that this gene is essential for de novo SA biosynthesis. Conversely, NANP knockout TF1 cells showed no α2,3 SA level change. iii) NANS knockout increases the susceptibility to the APC TF1 PIGA-null cells with NANS knockout (TF1 DKO) had significantly higher C5b-9 deposition when treated with NHS compared to deletion of either gene alone (Figure A), demonstrating the formation of membrane attack complex (MAC). Cell viability assays also showed that TF1 DKO cells had significantly higher complement-induced cell killing when treated with NHS. Both C5b9 deposition and killing were rescued by APC-specific inhibition targeting CFD, demonstrating SA biosynthesis defects will increase the susceptibility to the APC specifically. iv) NANS knockout decreases the binding capacity of CFH To investigate CFH binding to the cell surface, C3b was first evenly loaded onto cells, followed by the addition of recombinant CFH. NANS knockout cells displayed significantly reduced CFH binding capacity, providing a mechanism for APC activation in the NANS knockout. v) NANS M117I decreases de novo biosynthesis of SA To assess the NANS variant identified in a patient with aHUS, TF1 DKO cells were transduced with a lentivirus containing the either wild type or M117I-mutated NANS, and cell-sorted to select individual clones. After treatment with sialidase to remove all SA, cells with NANS M117I had significantly lower SA level recovery compared to NANS wild type cells (Figure B). Conclusion: Targeted sequencing of 34 patients with aHUS identified a germline NANS variant in one case, suggesting an association between aHUS and SA biosynthesis defects. Functional studies showed that NANS knockout and the NANS M117I variant decreased cell surface SA levels. Loss of NANS also caused a decrease in CFH binding capacity and uncontrolled APC activation with increased cell death. Disclosures Chaturvedi: Alexion: Honoraria, Membership on an entity's Board of Directors or advisory committees; Sanofi: Honoraria, Membership on an entity's Board of Directors or advisory committees.

Mouse Models of the Common, Recurring Type 1 von Willebrand Disease Mutations Y1584C and R1205H

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 22-22
Author(s):  
Cynthia M. Pruss ◽  
Mia Golder ◽  
Andrea Bryant ◽  
Erin Burnett ◽  
Kate Sponagle ◽  
...  
Keyword(s):  
Mouse Model ◽  
Knockout Mice ◽  
Knockout Mouse ◽  
Von Willebrand Disease ◽  
Wild Type ◽  
Thrombosis Model ◽  
The Common ◽  
Platelet Accumulation ◽  
Von Willebrand

Abstract Abstract 22 Introduction: Type 1 von Willebrand disease (VWD) is caused by mutations that result in moderate decreases in VWF antigen (VWF:Ag is 5–50% of normal levels) and a mild bleeding phenotype. The common recurrent VWF missense mutation Y1584C is associated with mildly decreased VWF:Ag levels, increased ADAMTS13 cleavage, as well as a possible increase in clearance. The Vicenza mutation, R1205H, exhibits a more severe phenotype (VWF:Ag ∼10%) and accelerated clearance. Although well described in patients and through in vitro studies, extensive controlled in vivo investigation of these mutations has yet to be performed. In this study, we compared both Y1584C and R1205H to wild type VWF using hydrodynamic gene delivery of mouse VWF and ADAMTS13 transgenes in the VWF knockout mouse to determine the pathological mechanisms associated with these variants. Methods: Hydrodynamic injections were performed using 100 μ g wild type (WT) or mutant mouse Vwf cDNA in Ringer's solution in 7–9 week old C57Bl6 VWF knockout mice, replacing plasma VWF. Co-injections with mouse Adamts13 cDNA were also performed. Mice were sampled at days 2, 5, 8, and then weekly. Mouse plasma was analyzed for complete blood counts, VWF:Ag, VWF propeptide, and VWF multimer structure. Thrombotic injury was induced using ferric chloride injury to the arterioles of the cremaster in VWF knockout mice expressing VWF:Ag levels from 0.5–2 U/ml. Platelets were labeled with Rhodamine-6G to evaluate platelet accumulation. Time to stable vessel occlusion and platelet accumulation by relative fluorescence intensity were compared. Results: Hydrodynamic injection caused no adverse events in any animals. Complete blood count values were unaffected for both variants compared to WT. Initial high VWF:Ag values at day 2 were similar for WT VWF (25.4 ± 2.5 U/ml, n= 12, mean U/ml±SEM, n) and Y1584C (26.8 ± 5.5, n= 10), but R1205H levels were 36% lower (16.3 ± 2.1, n= 10). Lower VWF:Ag levels were demonstrated in both “homozygous” and “heterozygous” forms for both type 1 mutations from days 14–42, when VWF expression plateaus. R1205H VWF:Ag was 34.3 ± 5.9% of WT (P < 0.001) and “heterozygous” 1:1 ratio R1205H/WT co-delivery was 27.5 ± 4.7% (p < 0.001). Y1584C was 29.4 ± 7.5% of WT (P < 0.001), and Y1584C/WT was 51.1 ± 4.6% (p < 0.001). VWF propeptide to VWF:Ag ratios (days 2–42) demonstrate that R1205H mouse VWF had an increased clearance rate (165.4 ± 13.5%, p < 0.001), while Y1584C was normal (97.1 ± 6.8 %, P > 0.05) compared to WT (100.0 ± 10.0%). The R1205H mutation showed no significant difference in multimer structure by mean multimer band numbers (days 2 to 42, 93 ± 16%, n = 4, P > 0.05) to wild type VWF (100 ± 12%, n = 4). In contrast, Y1584C had a significant decrease (66 ± 18%, n = 4, P < 0.001). This effect was exaggerated by co-delivery of mouse ADAMTS13 for Y1584C, but not R1205H. Y1584C showed reduced thrombus formation in a ferric chloride injury model while R1205H demonstrated similar thrombogenic activity to wild type VWF. Mean occlusion times were WT = 29.9 ± 2.1 minutes, n = 8, R1205H = 29.1 ± 4.0, n = 8 (p > 0.05), and Y1584C = 38.7 ± 1.1, n = 9 (p = 0.001). Total platelet accumulation was decreased for Y1584C (83.6 ± 6.3%, p = 0.043), but was similar for R1205H (103 ± 6.3, P = 0.72) and WT (100 ± 5.3%). Conclusions: This study demonstrates that these two type 1 VWD mutations have a strong observable effect in the VWF knockout mouse model. R1205H exhibits a large decrease in VWF:Ag levels and evidence of accelerated clearance with R1205H. However, there is no alteration in multimer structure and apparently normal participation in a thrombosis model. Y1584C, in contrast, shows a loss of high molecular weight multimers that is exacerbated by the additional expression of ADAMTS13, indicating that ADAMTS13 cleavage is increased. Y1584C also has an initially high VWF:Ag level that was less than WT levels from day 14 onward, but shows no alteration in clearance, suggesting that there is a biosynthetic defect. Y1584C shows a significant defect in the arteriolar thrombosis model, presenting a Type 2A VWD-like phenotype in the mouse model, which is more severe than the human phenotype. This study has elucidated several novel mechanistic details for these two mutations and highlights that the pathogenic aspects of type 1 VWD can be recapitulated in the VWF knockout hydrodynamic injection model. Disclosures: No relevant conflicts of interest to declare.

Platelet Activation and Aggregation Induced by Recombinant von Willebrand Factors Reproducing Four Type 2B von Willebrand Disease Missense Mutations

1998 ◽  
Vol 79 (01) ◽  
pp. 211-216 ◽  
Author(s):  
Lysiane Hilbert ◽  
Claudine Mazurier ◽  
Christophe de Romeuf
Keyword(s):  
Platelet Aggregation ◽  
Platelet Activation ◽  
Von Willebrand Disease ◽  
Platelet Glycoprotein ◽  
Missense Mutations ◽  
Inhibit Platelet Aggregation ◽  
Wild Type ◽  
A1 Domain ◽  
Von Willebrand ◽  
Willebrand Factor

SummaryType 2B of von Willebrand disease (vWD) refers to qualitative variants with increased affinity of von Willebrand factor (vWF) for platelet glycoprotein Ib (GPIb). All the mutations responsible for type 2B vWD have been located in the A1 domain of vWF. In this study, various recombinant von Willebrand factors (rvWF) reproducing four type 2B vWD missense mutations were compared to wild-type rvWF (WT-rvWF) for their spontaneous binding to platelets and their capacity to induce platelet activation and aggregation. Our data show that the multimeric pattern of each mutated rvWF is similar to that of WT-rvWF but the extent of spontaneous binding and the capacity to induce platelet activation and aggregation are more important for the R543Q and V553M mutations than for the L697V and A698V mutations. Both the binding of mutated rvWFs to platelets and platelet aggregation induced by type 2B rvWFs are inhibited by monoclonal anti-GPIb and anti-vWF antibodies, inhibitors of vWF binding to platelets in the presence of ristocetin, as well as by aurin tricarboxylic acid. On the other hand, EDTA and a monoclonal antibody directed against GPIIb/IIIa only inhibit platelet aggregation. Furthermore, the incubation of type 2B rvWFs with platelets, under stirring conditions, results in the decrease in high molecular weight vWF multimers in solution, the extent of which appears correlated with that of plasma vWF from type 2B vWD patients harboring the corresponding missense mutation. This study supports that the binding of different mutated type 2B vWFs onto platelet GPIb induces various degrees of platelet activation and aggregation and thus suggests that the phenotypic heterogeneity of type 2B vWD may be related to the nature and/or location of the causative point mutation.

Generation of a mouse model of Primary Hyperoxaluria Type 1 via CRISPR/Cas9 mediated gene editing

2019 ◽  
pp. 28-39
Author(s):  
Kimberly Coughlan ◽  
Rajanikanth Maganti ◽  
Andrea Frassetto ◽  
Christine DeAntonis ◽  
Meredith Wolfrom ◽  
...  
Keyword(s):  
Mouse Model ◽  
Gene Editing ◽  
Primary Hyperoxaluria ◽  
Primary Hyperoxaluria Type 1 ◽  
Primary Hyperoxaluria Type ◽  
Hyperoxaluria Type

[18F]-florbetaben PET/CT Imaging in the Alzheimer’s Disease Mouse Model APPswe/PS1dE9

2018 ◽  
Vol 16 (1) ◽  
pp. 49-55 ◽  
Author(s):  
J. Stenzel ◽  
C. Rühlmann ◽  
T. Lindner ◽  
S. Polei ◽  
S. Teipel ◽  
...  
Keyword(s):  
Alzheimer’S Disease ◽  
Alzheimer's Disease ◽  
Mouse Model ◽  
New Drugs ◽  
Imaging Data ◽  
Wild Type ◽  
Preclinical Research ◽  
Standardized Uptake Values ◽  
Pet Ct

Background: Positron-emission-tomography (PET) using 18F labeled florbetaben allows noninvasive in vivo-assessment of amyloid-beta (Aβ), a pathological hallmark of Alzheimer’s disease (AD). In preclinical research, [<sup>18</sup>F]-florbetaben-PET has already been used to test the amyloid-lowering potential of new drugs, both in humans and in transgenic models of cerebral amyloidosis. The aim of this study was to characterize the spatial pattern of cerebral uptake of [<sup>18</sup>F]-florbetaben in the APPswe/ PS1dE9 mouse model of AD in comparison to histologically determined number and size of cerebral Aβ plaques. Methods: Both, APPswe/PS1dE9 and wild type mice at an age of 12 months were investigated by smallanimal PET/CT after intravenous injection of [<sup>18</sup>F]-florbetaben. High-resolution magnetic resonance imaging data were used for quantification of the PET data by volume of interest analysis. The standardized uptake values (SUVs) of [<sup>18</sup>F]-florbetaben in vivo as well as post mortem cerebral Aβ plaque load in cortex, hippocampus and cerebellum were analyzed. Results: Visual inspection and SUVs revealed an increased cerebral uptake of [<sup>18</sup>F]-florbetaben in APPswe/ PS1dE9 mice compared with wild type mice especially in the cortex, the hippocampus and the cerebellum. However, SUV ratios (SUVRs) relative to cerebellum revealed only significant differences in the hippocampus between the APPswe/PS1dE9 and wild type mice but not in cortex; this differential effect may reflect the lower plaque area in the cortex than in the hippocampus as found in the histological analysis. Conclusion: The findings suggest that histopathological characteristics of Aβ plaque size and spatial distribution can be depicted in vivo using [<sup>18</sup>F]-florbetaben in the APPswe/PS1dE9 mouse model.

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