PLATELET FACTOR 8 AS A NOVEL THERAPY FOR HEMOPHILIA A Hemophilia A is the most common inherited severe bleeding diathesis and is due to a deficiency/dysfunction of coagulation Factor Vlll (F8). Replacement therapy is effective for bleeding episodes, but is expensive and remains associated with bleeding sequelae. Importantly, up to 30% of these patients develop antibodies that inhibit F8 activity. Strategies to treat bleeding complications in this subgroup of patients can be problematic, especially if immunotherapy/tolerance to eliminate the inhibitor is unsuccessful. We were the first to describe the ability of ectopically expressed B-domainless human (/?) F8 in platelets (p/7BF8) to be effective at correcting the bleeding diathesis in F8-deficient (F8null) mice. We also showed that phBF8 is 100-fold more effective than plasma hFQ in the face of circulating inhibitors. These advantages of phF8 are tempered by our observation that the resulting clots in p/?BF8/F8""" mice are less stable than clots formed in F8null mice after /7F8 infusions and embolize more easily. We therefore believe that phF8 may have clinical utility, but needs enhanced efficacy to improve its hemostatic function. There are 3 specific aims to this proposal:
Specific Aim 1 : Understand and improve clot formation in pF8/F8""" mice. We now present new findings since the prior submission that pF8 variants that increase specific activity and/or reduce the apoptotic effect of pF8 on megakaryopoiesis and/or increase pF8 expression levels can markedly improve hemostatic outcome in F8""" mice. Proposed studies will confirm these distinct processes and identify strong pF8 candidates to be tested in both a large animal F8""" model and in a human-mouse xenotransplant model as described below.
Specific Aim 2 : Large animal studies of pFS. The most effective pF8 variants defined in Specific Aim 1 will be studied using a lentiviral-based pF8 expression system to drive expression in the megakaryocytes of a canine F8"" model as a prelude to human studies. These canine studies will examine efficacy in improving spontaneous bleeding episodes and in bleeding models, including cuticular and FeCIa arterial and venous injury models, in these F8"" dogs.
Specific Aim 3 : Studies of pF8 in human hematopoietic cells. To address whether our lentiviral strategy will work in human hematopoietic cells, we propose to also examine lentiviral-delivery of the best pF8s into (3D34* human hematopoietic cells and demonstrate that we can achieve similar levels of F8 in human as in murine megakaryocytes and that the pF8 localizes within a-granules. Immune-compromised mice will also be reconstituted with these progenitor cells, and the level of F8 antigen and activity that can be achieved in platelets in these xenotransplanted mice measured. In summary, our prior studies have demonstrated that pF8 offers a novel therapeutic strategy for the care of patients with hemophilia A, especially for those patients with problematic circulating inhibitors, but this approach may also be associated with increased clot instability and embolization. The proposed studies will further analyze the features of F8 that can effect pF8 hemostatic efficacy in murine studies and carry these concepts and pF8 variants forward to large animal and pre-clinical human-mouse xenotransplant studies.
|Sauna, Zuben E; Lozier, Jay N; Kasper, Carol K et al. (2015) The intron-22-inverted F8 locus permits factor VIII synthesis: explanation for low inhibitor risk and a role for pharmacogenomics. Blood 125:223-8|
|Sullivan, Spencer K; Mills, Jason A; Koukouritaki, Sevasti B et al. (2014) High-level transgene expression in induced pluripotent stem cell-derived megakaryocytes: correction of Glanzmann thrombasthenia. Blood 123:753-7|
|Sherman, Alexandra; Schlachterman, Alexander; Cooper, Mario et al. (2014) Portal vein delivery of viral vectors for gene therapy for hemophilia. Methods Mol Biol 1114:413-26|
|Yazicioglu, Mustafa N; Monaldini, Luca; Chu, Kirk et al. (2013) Cellular localization and characterization of cytosolic binding partners for Gla domain-containing proteins PRRG4 and PRRG2. J Biol Chem 288:25908-14|
|Callejas, David; Mann, Christopher J; Ayuso, Eduard et al. (2013) Treatment of diabetes and long-term survival after insulin and glucokinase gene therapy. Diabetes 62:1718-29|
|Siner, Joshua I; Iacobelli, Nicholas P; Sabatino, Denise E et al. (2013) Minimal modification in the factor VIII B-domain sequence ameliorates the murine hemophilia A phenotype. Blood 121:4396-403|
|Mingozzi, Federico; High, Katherine A (2013) Immune responses to AAV vectors: overcoming barriers to successful gene therapy. Blood 122:23-36|
|Anguela, Xavier M; Sharma, Rajiv; Doyon, Yannick et al. (2013) Robust ZFN-mediated genome editing in adult hemophilic mice. Blood 122:3283-7|
|Buchlis, George; Podsakoff, Gregory M; Radu, Antonetta et al. (2012) Factor IX expression in skeletal muscle of a severe hemophilia B patient 10 years after AAV-mediated gene transfer. Blood 119:3038-41|
|Mingozzi, Federico; High, Katherine A (2011) Therapeutic in vivo gene transfer for genetic disease using AAV: progress and challenges. Nat Rev Genet 12:341-55|
Showing the most recent 10 out of 39 publications