In addition to their well-described roles in platelet adhesion and thrombus formation, many of the major human platelet membrane glycoproteins bear clinically-important alloantigenic determinants that can induce an alloimmune response in two well-described immunopathogenic syndromes: Post-transfusion purpura and neonatal alloimmune thrombocytopenia (NAIT). NAIT is estimated to complicate ~ 1 in 1000 pregnancies, and is caused by maternal antibodies generated in response to paternally-inherited antigens present on fetal platelets that re- cross the placenta and bind to fetal and/or neonatal platelets, resulting in thrombocytopenia often serious enough to require transfusion, and in the most severe cases causing intracranial hemorrhage and intrauterine death. Despite advances in treatment, NAIT remains the leading cause of intracranial hemorrhage in full-term infants, often leading to lifelong disability. Identification of the causative platelet-specific alloantibody in maternal sera is required to properly guide prenatal treatment, facilitate post-natal management, and manages future pregnancies; however current methods for their detection employ time-consuming, technically-demanding antigen-capture ELISA assays that require hundreds of microliters of maternal alloantisera. Despite decades of development, these methods fail to identify the offending alloantibody more than half the time because the target alloantigens (1) cannot be mimicked with linear peptides, (2) are often unstable when removed from its plasma membrane environment, and most importantly (3) are frequently not even available as targets in diagnostic laboratories be- cause individuals whose platelets express them are extremely rare and hard to come by. Taken together, there is a compelling need for transformative diagnostic platforms that can narrow the existing diagnostic gap to improve treatment and care of this important cause of newborn morbidity and mortality. In the present application, we propose to combine recent advances in CRISPR gene editing technology with the ability to generate megakaryocyte progenitor cells, megakaryocytes, and platelets from induced pluripotent stem cells to establish an entirely new platform for the field of Transfusion Medicine - namely the creation of platelet alloantigen-specific cell lines capable of long-term self-renewal, cryopreservation, and distribution, thereby providing a potentially inexhaustible source of iPS-derived platelets for diagnostic (and potentially future therapeutic) use. We also propose to exploit CRISPR technology to develop a novel humanized mouse model of NAIT that will allow us to resolve outstanding issues in platelet alloimmunity, including examining the hypothesis that certain subtypes of maternal anti-platelet alloantibodies are likely to result in severe NAIT and intracranial hemorrhage. Taking advantage of the opportunistic convergence of these new technologies has the potential to transform the field of platelet trans- fusion medicine in new and exciting ways that are both scientifically important and clinically beneficial.
Immune reactions to platelets, initiated either by transfusion or by pregnancy, are responsible for two serious immunopathogenic syndromes: Post-transfusion purpura and neonatal alloimmune thrombocytopenia. This grant application proposes novel studies that will lead to a greater understanding of the etiology of platelet alloimmune disorders, and will also develop novel reagents leading to greatly improved diagnostic testing.
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