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

生成用于诊断和研究用途的同种异体抗原特异性设计血小板

• 批准号: 9005358
• 负责人: Peter J Newman
• 金额: $ 51.05万
• 依托单位: VERSITI WISCONSIN, INC.
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-06-01 至 2020-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9005358
• 关键词: Adhesions; Alleles; Alloantigen; Amino Acids; Antibodies; Antibody Specificity; Antigen Targeting; Antigens; Applications Grants; Binding; Biological Assay; Blood Circulation; Blood Coagulation Disorders; Blood Platelets; CRISPR/Cas technology; Caring; Cell Line; Cell membrane; Cessation of life; Classification; Clinical; Clustered Regularly Interspaced Short Palindromic Repeats; Cryopreservation; DNA; Detection; Diagnosis; Diagnostic; Diagnostic tests; Disease; Environment; Enzyme-Linked Immunosorbent Assay; Epitopes; Etiology; Failure; Fetus; Frequencies; Future; Gene Frequency; Gene Pool; Generations; Genes; Genetic Polymorphism; Genotype; HLA Antigens; Hemorrhage; Human; Human Genome; Human Platelet Antigens; Immune; Immunology; Individual; Infant; Inherited; Intracranial Hemorrhages; Isoantibodies; Laboratories; Lead; Live Birth; Liver; Maternal antibody; Medicine; Megakaryocytes; Methods; Morbidity - disease rate; Mus; Neonatal; Neonatal Alloimmune Thrombocytopenia; Neonatal Thrombocytopenia; Newborn Infant; Nucleotides; Pathogenicity; Peptides; Placenta; Platelet Membrane Glycoproteins; Platelet Transfusion; Pregnancy; Production; Protein Isoforms; Purpura; Reaction; Reagent; Role; Severities; Source; Stem cells; Surface; Syndrome; System; Techniques; Technology; Therapeutic Uses; Thrombocytopenia; Thrombus; Time; Transfusion; Ursidae Family; base; clinical predictors; disability; fetal; humanized mouse; immunogenic; improved; induced pluripotent stem cell; isoimmunity; laboratory experience; macrophage; man; maternal serum; method development; mortality; mouse model; neonate; new technology; novel; prenatal; public health relevance; response; self-renewal; stem; time use

 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.

 描述（由申请人提供）：除了在血小板粘附和血栓形成中充分描述的作用外，许多主要的人类血小板膜糖蛋白还具有临床重要的同种抗原决定簇，可以在两种充分描述的免疫病理综合征中诱导同种免疫反应：输血后紫癍和新生儿同种免疫性血小板减少症（NAIT）。据估计，NAIT使约1/1000的妊娠复杂化，并且由母体抗体引起，所述母体抗体响应于存在于胎儿血小板上的父代遗传抗原而产生，所述父代遗传抗原重新穿过胎盘并结合至胎儿和/或新生儿血小板， 导致血小板减少症，通常严重到需要输血，并且在最严重的情况下导致颅内出血和子宫内死亡。尽管治疗取得了进展，但NAIT仍然是足月婴儿颅内出血的主要原因，通常导致终身残疾。鉴定母体血清中的致病性血小板特异性同种抗体是正确指导产前治疗、促进产后管理和管理未来妊娠所必需的;然而，目前用于其检测的方法采用耗时、技术要求高的抗原捕获ELISA测定，其需要数百微升的母体同种抗血清。尽管发展了几十年，但这些方法在一半以上的时间内不能鉴定出引起攻击的同种异体抗体，因为靶同种异体抗原（1）不能用线性肽模拟，（2）当从其质膜环境中除去时通常不稳定，最重要的是，（3）在诊断实验室中甚至常常不能作为目标，因为血小板能表达它们的个体非常罕见，很难找到。总之，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。在本申请中，我们提出将CRISPR基因编辑技术的联合收割机最新进展与从诱导多能干细胞产生巨核细胞祖细胞、巨核细胞和血小板的能力相结合，以建立用于输血医学领域的全新平台-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（以及潜在的未来治疗）用途提供了潜在的无限来源的iPS衍生血小板。我们还建议利用CRISPR技术开发一种新的NAIT人源化小鼠模型，这将使我们能够解决血小板同种异体免疫中悬而未决的问题，包括检查某些母体抗血小板同种异体抗体亚型可能导致严重NAIT和颅内出血的假设。利用这些新技术的机会性融合，有可能在新的和新的领域改变血小板输注医学。 既有科学意义又有临床效益的激动人心的方法。