GPI-ADAMTS13-Cultured Red Blood Cells

项目摘要/摘要 我们已经开发了PSC-RED，这是一种化学定义的可扩展方法，用于区分诱导多能性 干细胞(IPSCs)转化为去核培养的红细胞(CRBCs)，我们已经证明 可以产生表达GPI锚定的、截短的ADAMTS13片段的细胞，该片段能够有效地 当插入细胞膜时，切断其von Willebrand(VWF)同源识别位点。 这项建议的主要目的是测试输注几毫升治疗性GPI- ADAMTS13-cRBC可替代血浆置换治疗血栓性血小板减少症 紫癜症(TTP)。在目标1中，我们建议生产crbcs工程场地--特别是在AAVS1安全区-- 表达对自身抗体具有抵抗力的变异型GPI-ADAMTS13的港口站点 对特发性三叉神经痛负责。我们将验证这些GPI-ADAMTS13-cRBC对电池板的抗性 来自未经治疗的TTP患者的血浆，在体外使用一组 试验，并在体内使用基于注射氯屈膦酸盐脂质体的小鼠异种输血模型 (CloLip)和眼镜蛇毒素(CVF)，使人红细胞在小鼠体内存活多天 发行量。 在目标2中，我们将直接测试GPI-ADAMTS13红细胞(RBC)是否可以用于补偿 ADAMTS13在完全免疫能力的动物模型中失去活性。我们已经改造了一只老鼠 在ROSA26基因座的红细胞中特异性表达GPI-ADAMTS13。我们建议将这些特征描述为 在注射大量重组VWF的基础上，将它们输入TTP模型中 转化为ADAMTS13KO小鼠。如果成功，这些关键实验将提供一个原则证明 输注携带膜结合ADAMTS13的红细胞可用于治疗TTP。 我们已经证明，我们生产cRBC的方案可以用来区分橄榄狒狒的IPSCs 去核红细胞。在目标3中，我们建议在体外鉴定橄榄狒狒GPI-ADAMTS13-cRBC，并 测定IPSC来源的GPI-ADAMTS13-cRBC的体内半衰期和酶活性 动物模型。 工程crbcs是一种非常有前景的输血和药物转化研究途径。 交付字段。实现所提出的目标将为新的治疗方法提供临床前数据。 先天性和特发性TTP。拟议的实验还将验证一个强大的平台，以 生产和测试IPSC来源的治疗性cRBC，这可能有许多其他应用。

Project Summary/Abstract We have developed PSC-RED, a chemically-defined scalable method to differentiate induced pluripotent stem cells (iPSCs) into enucleated cultured Red Blood Cells (cRBCs) and we have demonstrated that we can generate cells expressing a GPI-anchored, truncated fragment of ADAMTS13 that is able to efficiently cleave its von Willebrand (VWF) cognate recognition site, while inserted in the cytoplasmic membrane. The main objective of this proposal is to test whether transfusion of a few mL of therapeutic GPI- ADAMTS13-cRBCs could replace plasma exchange as a treatment for Thrombotic Thrombocytopenic Purpura (TTP). In Aim 1, we propose to produce cRBCs engineered site-specifically at the AAVS1 safe- harbor site to express variant forms of GPI-ADAMTS13 that are resistant to the auto-antibodies responsible for idiopathic TTP. We will validate the resistance of these GPI-ADAMTS13-cRBCs to a panel of plasmas from untreated TTP patients, characterize their cellular properties, in vitro using a battery of tests, and in vivo using a murine xeno-transfusion models based on injection of clodronate liposomes (CloLip) and Cobra Venom Factor (CVF) that allows human RBCs to survive multiple days in the mouse circulation. In Aim 2, we will directly test whether GPI-ADAMTS13 red blood cells (RBCs) can be used to compensate ADAMTS13 loss of activity in a fully immuno-competent animal model. We have engineered a mouse that express GPI-ADAMTS13 specifically in RBCs from the rosa26 locus. We proposed to characterize these cells and to transfuse them in a model of TTP based on injection of large amounts of recombinant VWF into ADAMTS13KO mice. If successful, these key experiments will provide a proof-of-principle that transfusion of RBCs carrying a membrane-bound ADAMTS13 can be used as a treatment for TTP. We have shown that our protocol to produce cRBCs can be used to differentiate olive baboon iPSCs into enucleated cRBCs. In Aim 3, we propose to characterize olive baboon GPI-ADAMTS13-cRBCs in vitro, and to measure the half-life and the enzymatic activity of iPSC-derived GPI-ADAMTS13-cRBCs in vivo, in a large animal model. Engineered cRBCs are a highly promising avenue of translational research in the transfusion and the drug delivery fields. Accomplishing the proposed Aims will provide pre-clinical data for a novel treatment for congenital and idiopathic TTP. The proposed experiments will also validate a powerful platform to produce and test therapeutic iPSC-derived cRBCs which could have many other applications.