Engineering iPSC-RBCs for Transfusion

摘要：输血是住院患者最常见的治疗程序。 尽管使用标准供体来源的红细胞输血的疗效，我们认为， 输血的潜力还未被挖掘最近几个研究小组的研究表明，人类 诱导性多能干细胞（iPSC）可以来源于少量的外周血，可以经历 基因组编辑以产生精确的遗传变化，并可分化为终末成熟的RBC （iPSC-RBC），全部在符合cGMP的条件下。虽然iPSC-RBC可能无法取代供体来源的RBC， 在可预见的未来，常规输血，我们认为，iPSC-RBC已遗传 工程化以表达新功能（例如，预防镰状细胞病患者的同种免疫） 可以在目前输血疗法不足的地区找到近期的临床应用。在 适当的治疗利基，工程iPSC-RBC将是高价值的高影响力的产品，可以维持 由于其独特的特性，成本很高。调查小组建议利用最近的进展， iPSC和基因组编辑技术，以及他们正在进行的NHLBI资助的RBC生物学研究， 免疫学，追求3个具体目标： (1)优化cGMP级方案，用于人外周血淋巴细胞的建立、增殖和分化， 将血液来源的iPSC转化为成熟RBC（iPSC-RBC）; (2A)工程化iPSC，使得所得RBC对多种血型抗原呈阴性，和（2B） 表征输注时iPSC-RBC的体内存活、形态成熟和免疫原性 转化为专门的鼠输血模型;以及 (3)为了获得FDA IND和机构IRB批准，并进行iPSC的人自体输注- RBC，以研究受体中RBC的存活、成熟和免疫原性。 拟议的研究经过精心设计，以整合最近开发的技术（cGMP iPSC 方法，基因编辑）与专业能力在埃默里（EPIC cGMP洁净室在血库，在体内 生物素化RBC跟踪，首次在人类细胞治疗的专业知识），以研究人类iPSC的生物学- 在输注到人类受体后衍生的RBC。成功完成拟议的调查将 从而产生新型血液制品，以满足输血依赖患者重要的未满足的临床需求， 镰状细胞病患者

ABSTRACT: Blood transfusion is the most common therapeutic procedure performed for hospitalized patients. Despite the efficacy of transfusion using standard donor-derived RBCs, we believe the full therapeutic potential of blood transfusion remains untapped. Recent work from several groups has demonstrated that human induced pluripotent stem cells (iPSCs) can be derived from small volumes of peripheral blood, can undergo genome editing to produce precise genetic changes, and can be differentiated into terminally mature RBCs (iPSC-RBCs), all under cGMP-compliant conditions. While iPSC-RBCs may not replace donor-derived RBCs for routine transfusions in the foreseeable future, we believe that iPSC-RBCs that have been genetically engineered to express novel functionalities (eg, prevention of alloimmunization in sickle cell disease patients) could find near term clinical applications in areas where current transfusion therapies are inadequate. In the proper therapeutic niche, engineered iPSC-RBCs would be high-value high-impact products that could sustain high costs due to their unique characteristics. The investigative team proposes to leverage recent advances in iPSC and genomic editing technologies, as well their ongoing NHLBI-funded studies in RBC biology and immunology, to pursue 3 Specific Aims: (1) To optimize cGMP-grade protocols for establishment, propagation, and differentiation of human peripheral blood-derived iPSCs into mature RBCs (iPSC-RBCs); (2A) To engineer iPSCs such that resulting RBCs are negative for multiple blood group antigens, and (2B) To characterize the in vivo survival, morphological maturation, and immunogenicity of iPSC-RBCs when transfused into specialized murine transfusion models; and (3) To obtain FDA IND and institutional IRB approvals, and perform human autologous transfusions of iPSC- RBCs to investigate RBC survival, maturation, and immunogenicity in recipients. The proposed studies have been carefully designed to integrate recently developed techniques (cGMP iPSC methods, gene editing) with specialized capabilities at Emory (EPIC cGMP clean room in the blood bank, in vivo biotinylated RBC tracking, first-in-human cell therapy expertise) to investigate the biology of human iPSC- derived RBCs after transfusion into human recipients. Successful completion of the proposed investigations will lead to novel blood products to meet important unmet clinical needs in transfusion-dependent patients such as those with sickle cell disease.