Characterization of a genetically engineered mouse model of hemophilia A with complete deletion of the F8 gene

Chao, Brittany; Baldwin, W. Hunter; Healey, John F.; Parker, Ernest T.; Shafer‐Weaver, Kimberly; Cox, Courtney; Jiang, Ping; Kanellopoulou, Chrysi; Lollar, Pete; Meeks, Shannon L.; Lenardo, Michael J.

doi:10.1111/jth.13202

Cited by 12 publications

(16 citation statements)

References 43 publications

(56 reference statements)

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“…FVIII knockout mice (hemophilia A mice, TKO) on a C57BL/6 background were used for all experiments. 36 TKO mice possess a deletion of the entire F8 coding sequence 36 ; no F8 messenger RNA is detectable in these mice. TKO mice and E16 hemophilia A mice exhibit a similar bleeding phenotype as measured by factor activity and a tail-snip assay, and develop similar anti-FVIII antibody titers following recombinant FVIII exposure.…”

Section: Mice and Materialsmentioning

confidence: 98%

“…To more specifically test the role of MZ B cells in FVIII antibody formation, we next developed a protocol to deplete MZ B cells from hemophilia A mice. Given that multiple doses of FVIII are required for the development of a measurable anti-FVIII antibody response in this model, 36,42,43 and to mimic the administration schedule often used in hemophilia A patients, we sought to develop an approach that would enable MZ B-cell depletion throughout the first month of injection, thereby allowing for the first 4 weekly injections of FVIII to occur in the absence of MZ B cells. As MZ B cells only reside within the spleen in mice, parallel sampling of the spleen to determine MZ B-cell depletion efficacy and measurement of anti-FVIII antibody formation is not possible.…”

Section: Mz B-cell Depletion Prevents Fviii Inhibitor Formationmentioning

confidence: 99%

“…Following the initial round of FVIII administration, all mice received an additional double-dose "boost" of 4 mg of FVIII, as done previously. 36 Despite this higher dose exposure of FVIII, the majority of recipients depleted of MZ B cells during the primary exposure experienced a greatly reduced response. Indeed, the majority of MZ B-cell-depleted recipients completely failed to develop anti-FVIII antibodies (68%) or FVIII inhibitors (90%) when examined 7 days after administration of a "boosted" dose of FVIII ( Figure 3D-E), indicating variable MZ B-cell reconstitution at the time of FVIII boost.…”

Section: Mz B-cell Depletion Prevents Fviii Inhibitor Formationmentioning

confidence: 99%

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Marginal zone B cells are critical to factor VIII inhibitor formation in mice with hemophilia A

Zerra

Cox

Baldwin

et al. 2017

Blood

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Although factor VIII (FVIII) replacement therapy can be lifesaving for patients with hemophilia A, neutralizing alloantibodies to FVIII, known as inhibitors, develop in a significant number of patients and actively block FVIII activity, making bleeding difficult to control and prevent. Although a variety of downstream immune factors likely regulate inhibitor formation, the identification and subsequent targeting of key initiators in inhibitor development may provide an attractive approach to prevent inhibitor formation before amplification of the FVIII immune response occurs. As the initial steps in FVIII inhibitor development remain incompletely understood, we sought to define early regulators of FVIII inhibitor formation. Our results demonstrate that FVIII localizes in the marginal sinus of the spleen of FVIII-deficient mice shortly after injection, with significant colocalization with marginal zone (MZ) B cells. FVIII not only colocalizes with MZ B cells, but specific removal of MZ B cells also completely prevented inhibitor development following FVIII infusion. Subsequent rechallenge with FVIII following MZ B-cell reconstitution resulted in a primary antibody response, demonstrating that MZ B-cell depletion did not result in FVIII tolerance. Although recipient exposure to the viral-like adjuvant polyinosinic:polycytidylic acid enhanced anti-FVIII antibody formation, MZ B-cell depletion continued to display similar effectiveness in preventing inhibitor formation following FVIII infusion in this inflammatory setting. These data strongly suggest that MZ B cells play a critical role in initiating FVIII inhibitor formation and suggest a potential strategy to prevent anti-FVIII alloantibody formation in patients with hemophilia A.

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Section: Mice and Materialsmentioning

confidence: 98%

Section: Mz B-cell Depletion Prevents Fviii Inhibitor Formationmentioning

confidence: 99%

Section: Mz B-cell Depletion Prevents Fviii Inhibitor Formationmentioning

confidence: 99%

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Marginal zone B cells are critical to factor VIII inhibitor formation in mice with hemophilia A

Zerra

Cox

Baldwin

et al. 2017

Blood

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“…Bleeding was assessed in vivo, as described, 30 with a slight modification by transecting 3 mm instead of 4 mm of the distal tail and placing the proximal tail into a pre-warmed pre-weighed tube. Blood loss per gram of body weight was determined 40 min after the tail was clipped, or at the time of death.…”

Section: Tail Transectionmentioning

confidence: 99%

Gene Delivery of Activated Factor VII Using Alternative Adeno-Associated Virus Serotype Improves Hemostasis in Hemophiliac Mice with FVIII Inhibitors and Adeno-Associated Virus Neutralizing Antibodies

et al. 2017

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While therapeutic expression of coagulation factors from adeno-associated virus (AAV) vectors has been successfully achieved in patients with hemophilia, neutralizing antibodies to the vector and inhibitory antibodies to the transgene severely limit efficacy. Indeed, approximately 40% of mice transduced with human factor VIII using the AAV8 serotype developed inhibitory antibodies to factor VIII (FVIII inhibitor), as well as extremely high titers (≥1:500) of neutralizing antibodies to AAV8. To correct hemophilia in these mice, AAV9, a serotype with low in vitro cross-reactivity (≤1:5) to anti-AAV8, was used to deliver mouse-activated factor VII (mFVIIa). It was found that within 6 weeks of systemic administration of 2 × 10 particles/kg of AAV9/mFVIIa, hemophiliac mice with FVIII inhibitors and neutralizing antibodies (NAb) to AAV8 achieved hemostasis comparable to that in wild-type mice, as measured by rotational thromboelastometry. A level of 737 ng/mL mFVIIa was achieved after AAV9/mFVIIa adminstration compared to around 150 ng/mL without vector treatment, and concomitantly prothrombin time was shortened. Tissues collected after intra-articular hemorrhage from FVIII-deficient mice and mice with FVIII inhibitors were scored 4.7 and 5.5, respectively, on a scale of 0-10, indicating significant pathological damage. However, transduction with AAV9/mFVIIa decreased pathology scores to 3.6 and eliminated hemosiderin iron deposition in the synovium in most mice. Collectively, these results suggest that application of alternative serotypes of AAV vector to deliver bypassing reagents has the potential to correct hemophilia and prevent hemoarthrosis, even in the presence of FVIII inhibitor and neutralizing antibodies to AAV.

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“…The entire coding sequence was deleted by a Cre recombinase-LoxP site-mediated deletion. Plasma FVIII activity and anti-FVIII inhibitor titer induced after FVIII treatment were comparable to those of E16 mice [ 40 ].…”

Section: Reviewmentioning

confidence: 99%

Current animal models of hemophilia: the state of the art

et al. 2016

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Hemophilia is the most well-known hereditary bleeding disorder, with an incidence of one in every 5000 to 30,000 males worldwide. The disease is treated by infusion of protein products on demand and as prophylaxis. Although these therapies have been very successful, some challenging and unresolved tasks remain, such as reducing bleeding rates, presence of target joints and/or established joint damage, eliminating the development of inhibitors, and increasing the success rate of immune-tolerance induction (ITI). Many preclinical trials are carried out on animal models for hemophilia generated by the hemophilia research community, which in turn enable prospective clinical trials aiming to tackle these challenges. Suitable animal models are needed for greater advances in treating hemophilia, such as the development of better models for evaluation of the efficacy and safety of long-acting products, more powerful gene therapy vectors than are currently available, and successful ITI strategies. Mice, dogs, and pigs are the most commonly used animal models for hemophilia. With the advent of the nuclease method for genome editing, namely the CRISPR/Cas9 system, it is now possible to create animal models for hemophilia other than mice in a short period of time. This review presents currently available animal models for hemophilia, and discusses the importance of animal models for the development of better treatment options for hemophilia.

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Characterization of a genetically engineered mouse model of hemophilia A with complete deletion of the F8 gene

Cited by 12 publications

References 43 publications

Marginal zone B cells are critical to factor VIII inhibitor formation in mice with hemophilia A

Marginal zone B cells are critical to factor VIII inhibitor formation in mice with hemophilia A

Gene Delivery of Activated Factor VII Using Alternative Adeno-Associated Virus Serotype Improves Hemostasis in Hemophiliac Mice with FVIII Inhibitors and Adeno-Associated Virus Neutralizing Antibodies

Current animal models of hemophilia: the state of the art

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