Generation of alloantigen-specific Designer Platelets for diagnostic and investigative use

 描述(申请人提供)：除了它们在血小板黏附和血栓形成中的作用外，许多主要的人类血小板膜糖蛋白还带有临床上重要的同种异体抗原决定因素，可以在两种描述良好的免疫致病综合征中诱导同种免疫反应：输血后紫癜和新生儿同种免疫性血小板减少症(NAIT)。据估计，每1000例妊娠中就有1例并发NAIT，它是由母体抗体引起的，母体抗体是对胎儿血小板上存在的父系遗传抗原的反应而产生的，这些抗原重新穿过胎盘并与胎儿和/或新生儿血小板结合。 导致血小板减少，通常严重到需要输血，在最严重的情况下会导致颅内出血和宫内死亡。尽管在治疗方面取得了进展，NAIT仍然是足月儿颅内出血的主要原因，通常会导致终身残疾。为了正确指导产前治疗、促进产后管理和管理未来的怀孕，需要在孕妇血清中鉴定致病的血小板特异性同种抗体；然而，目前的检测方法使用耗时的、技术要求高的抗原捕获ELISA法，需要数百微升的孕妇同种抗体。尽管经过了几十年的发展，这些方法仍有一半以上的时间无法识别有害的同种异体抗体，因为目标同种异体抗原(1)不能与线性多肽模拟，(2)当从其质膜环境中移除时往往不稳定，最重要的是(3)经常不能作为诊断实验室的靶标，因为其血小板表达它们的个人极其罕见且很难获得。综上所述，迫切需要能够缩小现有“诊断差距”的变革性诊断平台，以改善对新生儿发病率和死亡率这一重要原因的治疗和护理。在目前的应用中，我们建议将CRISPR基因编辑技术的最新进展与从诱导的多能干细胞生成巨核祖细胞、巨核细胞和血小板的能力相结合，为输血医学领域建立一个全新的平台-即创建能够长期自我更新、冷冻保存和分布的血小板同种异体抗原特异性细胞系，从而为诊断(以及未来的治疗)提供潜在取之不尽的iPS来源的血小板。我们还建议利用CRISPR技术开发一种新的人源化NAIT小鼠模型，该模型将使我们能够解决血小板同种免疫中的突出问题，包括检验某些亚型的母体抗血小板同种抗体可能导致严重的NAIT和颅内出血的假说。利用这些新技术的机会性融合，有可能在新的和 令人兴奋的方法，既有重要的科学价值，也有临床上的益处。

 DESCRIPTION (provided by applicant): 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.