GPI-ADAMTS13-Cultured Red Blood Cells

项目概要/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，用于区分诱导的多能细胞， 我们已经证明，我们可以将干细胞（iPSC）转化为去核培养的红细胞（cRBC）， 可以产生表达ADAMTS13的GPI锚定的截短片段的细胞，该片段能够有效地 切割其von Willebrand（VWF）同源识别位点，同时插入细胞质膜。 本提案的主要目的是测试输注几mL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（TTP）。在目标1中，我们提出在AAVS1安全位点特异性地产生cRBC工程化， 表达抗自身抗体的GPI-ADAMTS13变体形式的港口位点 导致特发性TTP我们将验证这些GPI-ADAMTS13-cRBC对检测组的抗性 的血浆从未经治疗的TTP患者，表征其细胞特性，在体外使用电池 测试，并在体内使用基于注射氯膦酸盐脂质体的小鼠异种输血模型 （CloLip）和眼镜蛇毒因子（CVF），其允许人RBC在小鼠中存活多天 流通 在目标2中，我们将直接测试GPI-ADAMTS13红细胞（RBC）是否可用于补偿 在完全免疫活性动物模型中ADAMTS13活性丧失。我们设计了一只老鼠 在红细胞中特异性表达来自rosa26基因座的GPI-ADAMTS13。我们建议将这些 细胞，并将它们输注到基于注射大量重组VWF的TTP模型中 ADAMTS13KO小鼠。如果成功，这些关键实验将提供一个原理证明， 输注携带膜结合ADAMTS13的RBC可用作TTP的治疗。 我们已经表明，我们的产生cRBC的方案可以用于将橄榄狒狒iPSC分化为 去核cRBC。在目标3中，我们提出在体外表征橄榄狒狒GPI-ADAMTS13-cRBC，并且 为了在体内测量iPSC衍生的GPI-ADAMTS13-cRBC的半衰期和酶活性，在大的 动物模型 工程化cRBC是输血和药物转化研究的一个非常有前途的途径 交付字段。实现所提出的目标将为以下疾病的新型治疗提供临床前数据： 先天性和特发性TTP。拟议的实验还将验证一个强大的平台， 生产和测试治疗性iPSC衍生的cRBC，其可以具有许多其他应用。

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.