Universal Donor Megakaryocytes

通用供体巨核细胞

• 批准号: 9305130
• 负责人: THALIA STAMATOYANNOPOULOS
• 金额: $ 39.88万
• 依托单位: UNIVERSITY OF WASHINGTON
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-09-01 至 2019-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9305130
• 关键词: Affect; Allogenic; Antibodies; Antigens; Binding; Biological Markers; Bleeding time procedure; Blood Cells; Blood Platelets; CD4 Positive T Lymphocytes; CD8B1 gene; Cell Differentiation process; Cell Line; Cell Transplants; Cell surface; Cells; Chimeric Proteins; Clinic; Clinical; Clinical Trials; Clone Cells; Cytolysis; Dependovirus; Derivation procedure; Disease; Electron Microscopy; Engineering; Ensure; Flow Cytometry; Gene Expression; Gene Targeting; Genes; Genetic Polymorphism; Goals; Graft Rejection; HLA Antigens; Hemorrhage; Hepatitis B e Antigens; Histocompatibility Antigens Class I; Histocompatibility Antigens Class II; Histologic; Homologous Gene; Human; Immune; Immune response; Immunodeficient Mouse; In Vitro; Infusion procedures; KLRD1 gene; Knock-out; Label; Leukemic Cell; Life; Lung; MHC antigen; Measures; Mediating; Megakaryocytes; Modeling; Monitor; Morphology; Mouse Strains; Mus; Natural Killer Cells; Patients; Peripheral Blood Mononuclear Cell; Phase-Contrast Microscopy; Platelet Count measurement; Platelet Transfusion; Ploidies; Pluripotent Stem Cells; Production; Property; Proteins; Refractory; Regenerative Medicine; Research; SELP gene; Safety; Source; Stem cells; Surface; T-Lymphocyte; T-Lymphocyte and Natural Killer Cell; Tail; Testing; Thrombocytopenia; Time; Tissues; Transfusion; Transplantation; Veins; base; beta-2 Microglobulin; body system; cell bank; effective therapy; experimental study; human embryonic stem cell; human pluripotent stem cell; in vitro Assay; in vivo; induced pluripotent stem cell; nuclease; prevent; public health relevance; receptor; response; transcription factor; treatment response; vector

 DESCRIPTION (provided by applicant): Cellular products derived from pluripotent stem cells (PSCs) are being developed as possible treatments for diseases affecting almost every organ system. Megakaryocytes (MKs) are an especially promising application, since they produce platelets that are needed in thrombocytopenic patients to prevent bleeding, and they can be irradiated to ensure safety. However, the clinical use of cells derived from PSCs is limited by the host's rejection of transplanted cells due to polymorphisms in the human leukocyte antigen (HLA) genes. This has led to proposed solutions such as the establishment of HLA-typed stem cell banks or the derivation of PSCs from every patient, which will require that many different cell lines be established, characterized and approved for clinical use. Here we propose instead to develop universal donor PSCs derived by HLA- engineering that can be used in any recipient, which will greatly reduce the time and expense required to advance cellular therapies to the clinic. Our goal is to derive universal donor MKs from these PSCs that could be given to patients and produce platelets that function in the presence of the anti-HLA antibodies commonly found in transfusion-refractory patients. We will use adeno-associated virus (AAV) gene targeting vectors to knock out the B2M and RFXANK genes in human PSCs, which are required for HLA class I and II gene expression respectively. In order to prevent the lysis of HLA-negative cells by host Natural Killer (NK) cells, we will also express single chain versions of the non-polymorphic HLA-E class Ib protein fused to B2M, which should bind to the inhibitory CD94/NKG2A receptor on NK cells. These HLA-engineered PSCs will be differentiated in vitro into MKs and cultured with human blood cells to demonstrate that they no longer elicit immune responses or are lysed by NK cells. This approach will also be verified in mouse transplantation experiments by deriving analogous universal donor mouse PSCs, expressing the murine homolog of HLA-E (Qa-1b), differentiating these cells into MKs, and infusing the MKs into isogenic and allogeneic mouse recipients. These mouse experiments will directly test the hypothesis that universal donor MKs can survive in allogeneic hosts and produce functional platelets. We will also infuse human universal donor MKs into immunodeficient mice, to study the platelets they produce in vivo and the effects of anti-HLA antibodies.

 描述（由申请人提供）：来自多能干细胞（PSC）的细胞产品正在开发，作为影响几乎所有器官系统的疾病的可能治疗方法。巨核细胞（MK）是一个特别有前途的应用，因为它们产生血小板，血小板减少症患者需要防止出血，它们可以被照射，以确保安全。然而，衍生自PSC的细胞的临床应用受到限制 由于人类白细胞抗原（HLA）基因的多态性，宿主对移植细胞的排斥。这导致了提出的解决方案，例如建立HLA型干细胞库或从每个患者中衍生PSC，这将需要建立许多不同的细胞系，表征并批准用于临床应用。在这里，我们建议开发通过HLA工程衍生的通用供体PSC，其可以用于任何受体，这将大大减少将细胞疗法推进到临床所需的时间和费用。我们的目标是从这些PSC中获得通用供体MK，这些通用供体MK可以给予患者并产生在输血难治性患者中常见的抗HLA抗体存在下发挥作用的血小板。 我们将使用腺相关病毒（AAV）基因靶向载体敲除人PSC中的B2 M和RFXANK基因，这两个基因分别是HLA I类和II类基因表达所需的。为了防止HLA阴性细胞的溶解， 宿主自然杀伤（NK）细胞，我们还将表达与B2 M融合的非多态性HLA-E Ib类蛋白的单链形式，其应结合NK细胞上的抑制性CD 94/NKG 2A受体。这些HLA工程化的PSC将在体外分化为MK，并与人血细胞一起培养，以证明它们不再引起免疫应答或被NK细胞裂解。这种方法也将在小鼠移植实验中得到验证，通过衍生类似的通用供体小鼠PSC，表达HLA-E（Qa-1b）的鼠同源物，将这些细胞分化为MK，并将MK输注到同基因和同种异体小鼠受体中。这些小鼠实验将直接测试通用供体MK可以在同种异体宿主中存活并产生功能性血小板的假设。我们还将把人类通用供体MK输注到免疫缺陷小鼠中，以研究它们在体内产生的血小板和抗HLA抗体的作用。