Therapeutic Expression of a Platelet-Specific Integrin

血小板特异性整合素的治疗性表达

• 批准号: 6624447
• 负责人: DAVID Allen WILCOX
• 金额: $ 24.7万
• 依托单位: MEDICAL COLLEGE OF WISCONSIN
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-04-01 至 2006-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6624447
• 关键词: CD34 molecule; Retroviridae; SCID mouse; biological signal transduction; cardiovascular disorder therapy; complementary DNA; conformation; disease /disorder model; dogs; fibrinogen; gene expression; gene therapy; genetic promoter element; genetic transduction; glycoprotein biosynthesis; hematopoietic stem cells; hemostasis; immune tolerance /unresponsiveness; integrins; ligands; megakaryocytes; platelet activation; platelets; protein structure function; thrombasthenia; transfection /expression vector

During hemostasis, the primary response to injury is mediated by activation of the platelet-specific receptor, glycoproteins (GP) IIb-IIIa, which assumes a conformation capable of binding its ligand, fibrinogen. GPIIb-IIIa (alphaIIbbeta3) is a member of the integrin family of receptors that mediate the adhesion of cells to each other and to the extracellular matrix. GPIIb-IIIa is missing or defective in platelets of individuals having the autosomal recessive bleeding disorder, Glanzmann Thrombasthenia (GT). Molecular defects in the sequences encoding either GPIIb or GPIIIa prevent thrombasthenic platelets from binding fibrinogen and forming platelet aggregates. GT is characterized by lifelong mucocutaneous bleeding that can lead to severe intracranial or gastrointestinal bleeding resulting in death. Although platelet transfusions are used therapeutically, no cure exists for GT. My laboratory has recently developed methods for lineage-targeted gene therapy by utilizing the promoter of the human GPIIb gene to direct transgene expression in megakaryocytes derived from retroviral transduced hematopoietic CD34+ cells. As a result, we demonstrated correction of the GT phenotype in cultured megakaryocytes from human patients and beta3-null mice. This application proposes to test the hypothesis that retrovirus-mediated transduction of CD34+ cells with a lineage-specific promoter of the GPIIb gene will result in a sustained therapeutic level of transgene expression in megakaryocytes in vivo. The foundation of this plan rests on the observation that the GPIIb promoter targets proviral-derived transgene expression preferentially in megakaryocytes and thus an immune response to proviral-transduced stem cells should be avoided. Accordingly, a line of Great Pyrenees dogs affected with GT due to a defect in GPIIb will serve as an animal model to test the feasibility of lineage- targeted gene expression. Specifically, I propose to 1) Examine megakaryocyte-targeted GPIIb-IIIa synthesis and function following transduction of gene expression system, "pK9IIb", consisting of a retrovirus vector, the megakaryocyte-specific promoter of GPIIb, and cDNA encoding canine GPIIb into GT canine CD34+ hematopoietic cells, and 2) Transplant K9IIb-transduced GT canine CD34+ cells into immunocompromised mice and GT dogs to analyze the system's ability to correct the GT phenotype in platelets generated in these animals. This study should lay vital groundwork towards eventual lineage-targeted transgene expression with potential use for human gene therapy of hematological disorders.

在止血过程中，对损伤的主要反应是通过血小板特异性受体糖蛋白（GP）IIB-IIIA的激活介导的，该糖蛋白（GP）IIB-IIIA假定能够结合其配体纤维蛋白原的构象。 GPIIB-IIIA（alphaiibbeta3）是整合素受体家族的成员，可介导细胞彼此的粘附和细胞外基质。 GPIIB-IIIA在患有常染色体隐性出血障碍的个体的血小板中缺失或有缺陷，Glanzmann Thrombasthenia（GT）。 编码GPIIB或GPIIIA的序列中的分子缺陷可以防止血栓性血小板结合纤维蛋白原和形成血小板聚集体。 GT的特征是终身性粘膜性出血，可能导致严重的颅内或胃肠道出血，导致死亡。 尽管血小板输血是在治疗上使用的，但尚无治疗GT。 我的实验室最近通过利用人GPIIB基因的启动子来指导源自逆转录病毒转导的造血CD334+细胞的巨核细胞中的转基因表达来开发用于依赖谱系基因治疗的方法。 结果，我们证明了人类患者和beta3-null小鼠培养的巨核细胞中GT表型的校正。 该应用建议测试以下假设：逆转录病毒介导的CD34+细胞具有GPIIB基因的谱系特异性启动子的转导将导致体内巨核细胞中持续的转基因表达水平。 该计划的基础是基于这样的观察结果，即GPIIB启动子在巨核细胞中优先针对病毒衍生的转基因表达，因此应避免对前病毒转导的干细胞的免疫反应。 因此，由于GPIIB缺陷，一系列受GT影响的大比利牛斯犬将作为一种动物模型，以测试谱系靶向基因表达的可行性。 具体而言，我建议1）检查基因表达系统“ PK9IIB”，由逆转录病毒载体（由逆转录病毒载体）（由巨核细胞特定的gpiib的巨核细胞特异性促进剂）和c canne gpiib canine gpiib canine gpiib cyt34+ cantl cant34组成，检查基因表达系统“ PK9IIB”，检查巨核细胞靶向的GPIIB-IIIA合成和功能K9IIB转导的GT犬CD34+细胞进入免疫功能低下的小鼠和GT狗，以分析系统校正这些动物中血小板中GT表型的能力。 这项研究应为最终以谱系的转基因表达奠定重要的基础，并可能用于血液学疾病的人类基因治疗。