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Infused factor VIII-expressing platelets or megakaryocytes as a novel therapeutic strategy for hemophilia A.

ABSTRACT: B-domainless factor VIII (FVIII) ectopically expressed in megakaryocytes (MKs) is stored in ? granules of platelets (pFVIII) and is capable of restoring hemostasis in FVIII^null mice, even in the presence of circulating inhibitors. However, our prior studies have shown that this ectopically expressed pFVIII can injure developing MKs. Moreover, the known risks of prolonged thrombocytopenia after bone marrow transplantation are significant challenges to the use of this strategy to treat individuals with severe hemophilia A and particularly those with intractable clinically relevant inhibitors. Because of these limitations, we now propose the alternative therapeutic pFVIII strategy of infusing pFVIII-expressing MKs or platelets derived from induced pluripotent stem cells (iPSCs). pFVIII-expressing iPSC-derived MKs, termed iMKs, release platelets that can contribute to improved hemostasis in problematic inhibitor patients with hemophilia A. As proof of principle, we demonstrate that hemostasis can be achieved in vitro and in vivo with pFVIII-expressing platelets and show prolonged efficacy. Notably, pFVIII-expressing platelets are also effective in the presence of inhibitors, and their effect was enhanced with recombinant FVIIa. Human pFVIII-expressing iMKs improved hemostasis in vitro, and derived platelets from infused human pFVIII-expressing iMKs improved hemostasis in FVIII^null mice. These studies indicate the potential therapeutic use of recurrent pFVIII-expressing MK or platelet infusions with prolonged hemostatic coverage that may be additive with bypassing agents in hemophilia A patients with neutralizing inhibitors.

SUBMITTER: Lyde RB

PROVIDER: S-EPMC6517653 | biostudies-literature | 2019 May

REPOSITORIES: biostudies-literature

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Infused factor VIII-expressing platelets or megakaryocytes as a novel therapeutic strategy for hemophilia A.

Lyde Randolph B RB Ahn Hyun Sook HS Vo Karen K KK Jarocha Danuta J DJ Tkaczynski John J Treffeisen Elsa E Sullivan Spencer K SK Camire Rodney M RM Sabatino Denise E DE French Deborah L DL Poncz Mortimer M

Blood advances 20190501 9

B-domainless factor VIII (FVIII) ectopically expressed in megakaryocytes (MKs) is stored in α granules of platelets (pFVIII) and is capable of restoring hemostasis in FVIII<sup>null</sup> mice, even in the presence of circulating inhibitors. However, our prior studies have shown that this ectopically expressed pFVIII can injure developing MKs. Moreover, the known risks of prolonged thrombocytopenia after bone marrow transplantation are significant challenges to the use of this strategy to treat ...[more]

PMID: 31036722

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Project description:Essentials Platelet-Factor (F) VIII gene therapy is a promising treatment in hemophilia A. This study aims to evaluate if platelet-FVIII expression would increase the risk for thrombosis. Targeting FVIII expression to platelets does not induce or elevate thrombosis risk. Platelets expressing FVIII are neither hyper-activated nor hyper-responsive. SUMMARY:Background Targeting factor (F) VIII 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 play important roles not only in hemostasis, but also in thrombosis and inflammation. Objective To evaluate whether platelet-FVIII expression might increase thrombotic risk and thereby compromise the safety of this approach. Methods In this study, platelet-FVIII-expressing transgenic mice were examined either in steady-state conditions or under prothrombotic conditions induced by inflammation or the FV Leiden mutation. Native whole blood thrombin generation assay, rotational thromboelastometry analysis and ferric chloride-induced vessel injury were used to evaluate the hemostatic properties. Various parameters associated with thrombosis risk, including D-dimer, thrombin-antithrombin 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 levels of platelet-expressed FVIII than are therapeutically required to restore hemostasis in hemophilic mice. Under both steady-state conditions and prothrombotic conditions induced by lipopolysaccharide-mediated inflammation or the FV Leiden mutation, supratherapeutic levels of platelet-expressed FVIII did not appear to be thrombogenic. Furthermore, FVIII-expressing platelets were neither hyperactivated nor hyperactivatable upon agonist activation. Conclusion We conclude that, in mice, more than 30-fold higher levels of platelet-expressed FVIII than are required for therapeutic efficacy in hemophilia A are not associated with a thrombotic predilection.

Project description:BackgroundThe goal of gene therapy for patients with hemophilia A is to safely impart long-term stable factor VIII expression that predictably ameliorates bleeding with the use of the lowest possible vector dose.MethodsIn this phase 1-2 trial, we infused an investigational adeno-associated viral (AAV) vector (SPK-8011) for hepatocyte expression of factor VIII in 18 men with hemophilia A. Four dose cohorts were enrolled; the lowest-dose cohort received a dose of 5 × 1011 vector genomes (vg) per kilogram of body weight, and the highest-dose cohort received 2 × 1012 vg per kilogram. Some participants received glucocorticoids within 52 weeks after vector administration either to prevent or to treat a presumed AAV capsid immune response. Trial objectives included evaluation of the safety and preliminary efficacy of SPK-8011 and of the expression and durability of factor VIII.ResultsThe median safety observation period was 36.6 months (range, 5.5 to 50.3). A total of 33 treatment-related adverse events occurred in 8 participants; 17 events were vector-related, including 1 serious adverse event, and 16 were glucocorticoid-related. Two participants lost all factor VIII expression because of an anti-AAV capsid cellular immune response that was not sensitive to immune suppression. In the remaining 16 participants, factor VIII expression was maintained; 12 of these participants were followed for more than 2 years, and a one-stage factor VIII assay showed no apparent decrease in factor VIII activity over time (mean [±SD] factor VIII activity, 12.9±6.9% of the normal value at 26 to 52 weeks when the participants were not receiving glucocorticoids vs. 12.0±7.1% of the normal value at >52 weeks after vector administration; 95% confidence interval [CI], -2.4 to 0.6 for the difference between matched pairs). The participants had a 91.5% reduction (95% CI, 88.8 to 94.1) in the annualized bleeding rate (median rate, 8.5 events per year [range, 0 to 43.0] before vector administration vs. 0.3 events per year [range, 0 to 6.5] after vector administration).ConclusionsSustained factor VIII expression in 16 of 18 participants who received SPK-8011 permitted discontinuation of prophylaxis and a reduction in bleeding episodes. No major safety concerns were reported. (Funded by Spark Therapeutics and the National Heart, Lung, and Blood Institute; ClinicalTrials.gov numbers, NCT03003533 and NCT03432520.).

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Infused factor VIII-expressing platelets or megakaryocytes as a novel therapeutic strategy for hemophilia A.

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Infused factor VIII-expressing platelets or megakaryocytes as a novel therapeutic strategy for hemophilia A.

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