Generation of cultured RBCs with rare phenotypes for transfusion from sources usually discarded during regular blood donations

摘要 在发达国家，接受同种免疫的患者通常无法获得稀有表型的血液。对.的使用 培养的红细胞(CRBC)以满足这些临床需求获得了势头，当我们的实验室，包括其他实验室， 规模化生产碳氢化合物的发达条件。目前，包括我们在内的几个实验室 解决将cRBC作为临床产品的众多障碍。 2011年，杜伊实验室证明，2.5x1010自体红细胞(5毫升血液)从 动员后的CD34pos细胞在体内能正常存活。然而，这项开创性的研究有四个警告：1) 动员不太可能被接受为产生用于临床或诊断用途的红细胞的程序，2) 产生的碳红细胞量(约5毫升血液)比输血所需的最低剂量低一对数 儿童(50毫升或2.5×1011红细胞)，3)正常志愿者不是第一次临床试验评估的理想接受者 4)生产CRBC的高成本限制了它们作为产品的临床应用。 这项提案将通过提供临床上有用的crbcs剂量的原则证明来解决这些警告。 可以从废弃的干细胞来源中产生，用于异体免疫患者的输血 在没有替代品的情况下，生产碳红细胞的成本被认为是合理的，这是罕见的表型。 这一建议是基于基础科学家(Migliaccio博士和Bieker，Icahn)之间的密切合作 医学院和西奈山医学院)和临床医生(克莱恩博士和弗莱格尔博士，输血医学部 美国国家卫生研究院)。这些研究人员将进行实验，以确定是否通过- 目前从常规献血中丢弃的稀有表型的产品适合产生 2.5x1011 cRBC(目标1)使用基于“基因编辑”的新培养策略，将增加产量，同时 降低生产成本(目标2)。 我们认为，Migliaccio博士和Bieker博士以及两人之间之前的合作记录 新的合作者(Funnel和Zon博士)，就与这一应用和Long有关的科学问题 Klein和Flegel博士对镰状细胞性贫血患者临床治疗的长期兴趣，包括 具有罕见的表型，确保了这项提议的科学和临床方面的协同发展。 这是最大限度地有效地生产足够数量的适合的cRBC的可能性所必需的。 进行首例人类同种异体输血。

Abstract In developed countries, blood with rare phenotypes for alloimmunized patients is often unavailable. The use of cultured RBCs (cRBCs) to address these clinical needs gained momentum when our laboratory, among others, developed conditions for large scale production of cRBCs. Presently, several laboratories, including ours, are addressing the numerous barriers to establish cRBCs as clinical product. In 2011, the Douay laboratory demonstrated that 2.5x1010 autologous cRBCs (5 mL of blood) generated from mobilized CD34pos cells have normal survival in vivo. However, this ground breaking study has four caveats: 1) mobilization is unlikely to be accepted as procedure to generate cRBCs for clinical or diagnostic use, 2) the amount of cRBCs generated (~5 mL of blood) is one-log lower than the minimal dose required to transfuse a child (50 mL or 2.5x1011 RBCs), 3) normal volunteers are not ideal recipients for the first clinical trial to assess efficacy, and 4) the high costs to produce the cRBC limit their clinical use as product. This proposal will address these caveats by providing proof-of-principle that clinically useful doses of cRBCs can be generated from discarded stem cell sources and used for transfusion of alloimmunized patients with rare phenotypes for whom, having no alternatives, the costs to produce cRBCs is considered reasonable. This proposal is based on a close collaboration among basic scientists (Drs. Migliaccio and Bieker, Icahn School of Medicine and Mount Sinai) and clinicians (Drs. Klein and Flegel, Transfusion Medicine Department of the NIH). These investigators will perform experiments aimed to establish whether leukoreduction by- products currently discarded from regular blood donations with rare phenotypes are suitable to generate 2.5x1011 cRBCs (Aim 1) using novel culture strategies based on “gene editing” that will increase yields while reducing production costs (Aim 2). We believe that the documented previous colloboration between Drs Migliaccio and Bieker, as well as the two new collaborators (Drs. Funnel and Zon), on scientific questions related to this application and the long standing interest of Drs Klein and Flegel in clinical management of Sickle Cell Anemia patients, including those with rare phenotypes, assures the synergistic development of scientific and clinical aspects of this proposal. This is necessary to maximize the likelihood of efficiently producing adequate numbers of cRBCs well-suited for the first-in-man allogenic transfusion.