DOWNREGULATION OF HLA CLASS II ANTIGENS BY ANTISENSE DNA

反义 DNA 下调 HLA II 类抗原

• 批准号: 3747084
• 负责人: SARLA PUROHIT
• 金额: --
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/3747084
• 关键词: MHC class II antigen; Macaca mulatta; Retroviridae; antisense nucleic acid; bone marrow transplantation; dihydrofolate reductase; electroporation; flow cytometry; gene expression; genetic manipulation; genetic markers; human tissue; immune response genes; immune tolerance /unresponsiveness; immunoregulation; mixed lymphocyte reaction test; molecular cloning; monoclonal antibody; tissue /cell culture; transfection; transposon /insertion element

Class II Human Leukocyte Antigens (HLA) regulate the overall immune response and also trigger the rejection of a transplanted organ. The specific aim of the proposed research is to downregulate the expression of HLA class II antigens (HLA) . The inhibition of expression of HLA class II genes will be achieved by transfecting cells with the recombinant molecules containing the cloned cDNA from the D region of the HLA complex in antisense orientation. These constructs will have the dominant selectable markers, hph or neo for selection or the mutant dihydrofolate reductase (DHFR*) gene for selection and amplification of the transgenomic fragments in mammalian cells. Expression vectors, episomal replicons and retroviral vectors will be used for the construction of the recombinant molecules. The molecules containing the insert will be transferred into the cells by electroporation in case of the expression vectors and episomal vectors and by infection when the retrovirus vectors are used as the means of gene transfer. Reduction in cell surface expression of class II antigens on the drug resistant cell lines will be evaluated by monoclonal antibody staining with flow cytometric determinations and by the ability to activate cell proliferation in the mixed lymphocyte culture. The long-term goal of the proposed project is to apply the technique of antisense cDNA to the downregulation of the expression of HLA class II antigens in bone marrow cells and to induce tolerance to transplantation of mismatched and haplocompatible bone marrow stem cells to the recipient. Non-human primates will be used for preclinical trials.

II 类人类白细胞抗原 (HLA) 调节整体免疫 反应并引发移植器官的排斥反应。这 拟议研究的具体目的是下调表达 HLA II 类抗原 (HLA) 。抑制HLA II类的表达 基因将通过用重组分子转染细胞来获得 含有来自 HLA 复合物 D 区的克隆 cDNA 反义方向。这些构建体将具有主导选择 标记物，用于选择的 hph 或 neo 或突变的二氢叶酸还原酶 (DHFR*) 基因用于选择和扩增转基因片段 在哺乳动物细胞中。表达载体、附加型复制子和逆转录病毒 载体将用于构建重组分子。这 含有插入片段的分子将通过以下方式转移到细胞中： 对于表达载体和附加型载体，电穿孔和 当使用逆转录病毒载体作为基因手段时通过感染 转移。 II类抗原的细胞表面表达减少 通过单克隆抗体染色评估耐药细胞系 通过流式细胞术测定和激活细胞的能力 混合淋巴细胞培养物中的增殖。该组织的长期目标 拟议的项目是将反义 cDNA 技术应用于 骨髓中HLA II类抗原表达的下调 细胞并诱导对错配和移植的耐受性 将单倍体相容的骨髓干细胞输送给受体。非人类灵长类动物 将用于临床前试验。