GPI-ADAMTS13-Cultured Red Blood Cells

GPI-ADAMTS13-培养的红细胞

• 批准号: 10645340
• 负责人: ERIC E BOUHASSIRA
• 金额: $ 8.96万
• 依托单位: ALBERT EINSTEIN COLLEGE OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-09-15 至 2024-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10645340
• 关键词: Animal Model; Autoantibodies; Autoimmune Diseases; Autologous; Binding; Biological Assay; Blood; Blood Circulation; Cell Therapy; Cell membrane; Cells; Chemicals; Clinical Data; Clinical Trials; Coagulation Process; Dichloromethylene Diphosphonate; Disease; Drug Delivery Systems; Engineering; Erythrocyte Transfusion; Erythrocytes; Flow Cytometry; Genetic Engineering; Half-Life; Harvest; Hemoglobin; Human; Immunocompetent; In Vitro; Infusion procedures; Injections; Life; Liposomes; Long-Term Effects; Measurement; Measures; Membrane; Methods; Minor; Modeling; Modification; Mus; Oxygen; Papio; Papio anubis; Patients; Pharmaceutical Preparations; Plasma; Plasma Exchange; Production; Property; Protocols documentation; Recombinants; Relapse; Resistance; Savings; Site; Stream; Testing; Therapeutic; Thrombotic Thrombocytopenic Purpura; Toxic effect; Transfusion; Transgenic Mice; Transgenic Model; Translational Research; Variant; base; cellular engineering; cobra venom factor; enzyme activity; experimental study; in vivo; induced pluripotent stem cell; milliliter; mortality; mouse model; novel; overexpression; pre-clinical; stem cells; therapeutic evaluation; treatment choice

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.

项目摘要/摘要 我们已经开发了PSC-RED，一种化学定义的可伸缩方法来区分引起的多能 干细胞（IPSC）进入含蛋白培养的红细胞（CRBC）中，我们已经证明了我们 可以生成表达ADAMTS13的GPI锚定的截断片段的单元格，该片段能够有效 在插入细胞质膜的同时，将其von Willebrand（VWF）同源识别位点切割。 该提案的主要目的是测试是否输血了几毫升的治疗性GPI- ADAMTS13-CRBC可以代替血浆交换作为血栓性血小板减少性的治疗 紫癜（TTP）。在AIM 1中，我们建议在AAVS1 Safe- 表达对自动抗体具有抵抗力的GPI-ADAMTS13变体形式的港口。 负责特发性TTP。我们将验证这些GPI-ADAMTS13-CRBC对面板的阻力 未经治疗的TTP患者的等离子体，用一组电池在体外表征其细胞特性 测试，并使用基于注射氯膦酸盐脂质体的鼠异种转移模型进行体内 （Clolip）和眼镜蛇毒液因子（CVF），允许人RBC在小鼠中生存多天 循环。 在AIM 2中，我们将直接测试GPI-ADAMTS13红细胞（RBC）是否可用于补偿 ADAMTS13完全免疫能力的动物模型中的活性丧失。我们已经设计了一只鼠标 Express GPI-Adamts13在ROSA26基因座的RBC中专门在RBC中。我们建议将这些特征 细胞并根据注入大量重组VWF的TTP模型将它们输送 进入ADAMTS13KO小鼠。如果成功，这些关键实验将提供原则上的证明 携带膜结合ADAMTS13的RBC的输血可用作TTP的治疗方法。 我们已经表明，生产CRBC的协议可用于将橄榄狒狒IPSC区分开 浓缩的CRBC。在AIM 3中，我们建议在体外表征Olive Baboon GPi-Adamts13-Crbcs，并且 在体内测量IPSC衍生的GPI-ADAMTS13-CRBC的半衰期和酶活性， 动物模型。 工程化的CRBC是输血和药物转化研究的备受希望的途径 送货场。完成提议的目标将为临床前数据提供新的治疗方法 先天性和特发性TTP。拟议的实验还将验证一个强大的平台 生产和测试具有许多其他应用的IPSC衍生的CRBC。