EVALUATING THE ENGINEERING OF CYTOMEGALOVIRUS-SPECIFIC CD8+ T CELL CLONES

评估巨细胞病毒特异性 CD8 T 细胞克隆的工程设计

• 批准号: 8172786
• 负责人: STANLEY R. RIDDELL
• 金额: $ 15.51万
• 依托单位: UNIVERSITY OF WASHINGTON
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-05-01 至 2011-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8172786
• 关键词: Adoptive Immunotherapy; Adoptive Transfer; Adrenal Cortex Hormones; Allogenic; Antigens; Autologous; CD8B1 gene; Cell Therapy; Clone Cells; Cloning; Computer Retrieval of Information on Scientific Projects Database; Cytomegalovirus; Engineering; Exhibits; Funding; Glucocorticoid Receptor; Graft Rejection; Grant; Hematopoietic stem cells; Immunity; Immunocompetent; Immunosuppressive Agents; Immunotherapy; In Vitro; Institution; Macaca; Mature T-Lymphocyte; Modeling; Morbidity - disease rate; Organ Transplantation; Patients; Receptor Gene; Receptor Signaling; Research; Research Personnel; Resistance; Resources; Safety; Solid; Source; T cell response; T-Lymphocyte; United States National Institutes of Health; Virus Diseases; Zinc Fingers; endonuclease; graft vs host disease; immunosuppressed; innovation; mortality; peripheral blood; receptor; vector

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Reactivation of cytomegalovirus (CMV) represents a major cause of morbidity and mortality in immunosuppressed recipients of allogeneic hematopoietic stem cell and solid organ transplant as a consequence of deficient CD8+ T cell immunity. Adoptive immunotherapy with antigen-specific T cell clones has been shown to safely correct quantitative or qualitative deficiencies of T cells that permit progression of viral infection. However, T cell therapy has failed to restore protective T cell immunity in the subset of patients that receive immunosuppressive corticosteroids to treat graft versus host disease or graft rejection, respectively. To overcome this obstacle, we have investigated an innovative strategy for selectively interfering with glucocorticoid receptor (GR) signaling using zinc-finger endonucleases (ZFNs) for targeted deletion of the GR gene in mature T cells. We have developed a macaque model for adoptive transfer of CMV-specific T cell clones and employ this model to investigate the safety and efficacy of immunotherapy with CMV-specific T cells that lack the GR gene (GR-/-) after ZFN targeted deletion. We identified immunocompetent macaques with detectable CMV-specific CD8+ T cell responses in the peripheral blood and used a replication defective Ad5/35 vector to deliver ZFNs that target the GR gene in macaque T cells. CMV-specific GR-/- CD8+ T cell clones were then isolated by limiting dilution cloning. The GR-/- T cell clones exhibited CMV-specific cytolytic reactivity comparable to CMV-specific GRwt clones. However, in contrast to GRwt T cells, the GR-/- T cells were resistant to lymphotoxic effects of corticosteroids in vitro, consistent with disruption of the GR gene and lack of a functional GR protein. We are currently generating autologous GR-/- CMV-specific macaque CD8+ T cell clones from 2 macaques that can be used for adoptive transfer studies to evaluate the safety and efficacy of adoptively transferred CD8+ CMV-specific T cell clones with an edited GR gene.

该子项目是利用该技术的众多研究子项目之一 资源由 NIH/NCRR 资助的中心拨款提供。子项目和 研究者 (PI) 可能已从 NIH 的另一个来源获得主要资金， 因此可以在其他 CRISP 条目中表示。列出的机构是 对于中心来说，它不一定是研究者的机构。 由于 CD8+ T 细胞免疫缺陷，巨细胞病毒 (CMV) 的再激活是同种异体造血干细胞和实体器官移植的免疫抑制受者发病和死亡的主要原因。使用抗原特异性 T 细胞克隆的过继免疫疗法已被证明可以安全地纠正导致病毒感染进展的 T 细胞的定量或定性缺陷。然而，在接受免疫抑制性皮质类固醇治疗移植物抗宿主病或移植物排斥反应的患者中，T 细胞疗法未能恢复保护性 T 细胞免疫。为了克服这一障碍，我们研究了一种创新策略，使用锌指核酸内切酶 (ZFN) 选择性干扰糖皮质激素受体 (GR) 信号传导，从而靶向删除成熟 T 细胞中的 GR 基因。我们开发了一种用于 CMV 特异性 T 细胞克隆过继转移的猕猴模型，并利用该模型来研究 ZFN 靶向删除后缺乏 GR 基因（GR-/-）的 CMV 特异性 T 细胞免疫治疗的安全性和有效性。我们鉴定出具有免疫活性的猕猴，其外周血中具有可检测到的 CMV 特异性 CD8+ T 细胞反应，并使用复制缺陷型 Ad5/35 载体来递送针对猕猴 T 细胞中 GR 基因的 ZFN。然后通过有限稀释克隆分离 CMV 特异性 GR-/- CD8+ T 细胞克隆。 GR-/- T 细胞克隆表现出与 CMV 特异性 GRwt 克隆相当的 CMV 特异性细胞溶解反应性。然而，与 GRwt T 细胞相比，GR-/- T 细胞在体外对皮质类固醇的淋巴毒性作用具有抵抗力，这与 GR 基因的破坏和功能性 GR 蛋白的缺乏一致。我们目前正在从 2 只猕猴中生成自体 GR-/- CMV 特异性猕猴 CD8+ T 细胞克隆，可用于过继转移研究，以评估具有编辑的 GR 基因的过继转移 CD8+ CMV 特异性 T 细胞克隆的安全性和有效性。