LEUKOCYTE ADHERENCE DEFICIENCY MODEL FOR STEM CELL TRANSDUCTION

干细胞转导的白细胞粘附缺陷模型

• 批准号: 6110536
• 负责人: DENNIS DURAND HICKSTEIN
• 金额: $ 20.18万
• 依托单位: FRED HUTCHINSON CANCER RESEARCH CENTER
• 依托单位国家: 美国
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-09-01 至 1999-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6110536
• 关键词: CD antigens; Retroviridae; autologous transplantation; biotechnology; cell transformation; colony stimulating factor; flow cytometry; gene expression; gene therapy; genetic transduction; hematopoietic growth factor; hematopoietic stem cells; human tissue; integrins; leukapheresis; leukocyte adhesion molecules; leukocyte disorder; mixed tissue /cell culture; model design /development; tissue /cell culture; transfection /expression vector

This project aims to develop leukocyte adherence deficiency (LAD) as a model for applying advances in understanding of stem cell biology to transduction of the hematopoietic stem cell. LAD is characterized clinically by recurrent, life-threatening, bacterial infections due to the inability of leukocytes to adhere to the vessel wall and migrate to the site of infection. These adherence defects stem from the inability of leukocytes from affected children to express the CD11/CD18 leukocyte integrin heterodimers on the leukocyte surface due to primary genetic defects in the CD18 subunit. Leukocyte adherence deficiency is an attractive model for the proposed studies for several reasons: 1) the disease is life-threatening in the severe deficiency form, and except for bone marrow transplantation there is no treatment other than supportive therapy; 2) the disease is due to a defect in a single gene, the CD18 subunit, and the gene has been cloned; 3) transduction of a normal CD 18 gene into LAD EBV B cells has been shown to correct the biochemical and functional defect; 4) results from bone marrow transplantation indicate that the defect resides in the hematopoietic stem cell; 5) based upon gene expression by leukocytes in the moderate deficiency phenotype of LAD, low levels of CD11/CD18 expression appear to be sufficient to correct the severe clinical manifestations of the disease; and 6) since the defect involves a membrane receptor, a quantitative assessment of gene transfer and expression can be accomplished using flow cytometry of peripheral blood leukocytes. In the current studies we will build on our preliminary data identifying retroviral vectors, packaging cell lines, and culture conditions facilitating transduction of long term culture initiating cells. The specific aims of this project are: 1) to determine the conditions and vectors required for effective transduction of long term culture initiating cells; 2) to determine the ability of peripheral blood stem cells from children with LAD to be transduced using ex vivo retroviral- mediated gene transduction of the leukocyte integrin CD 18 subunit followed by reinfusion of the transduced cells; ,and 3) to determine if prior administration of a conditioning regimen enhances expression of the leukocyte integrin CD18 subunit in leukocytes following ex vivo transduction of CD 18 into peripheral blood stem cells from children with LAD. We anticipate that these studies will incorporate advances in understanding of the hematopoietic stem cell from the other projects in the SCOR, including those identifying ways of eliciting or selecting stem cells that are most susceptible to transduction, and those developing vectors and culture conditions supporting transduction of hematopoietic stem cells. The results of these studies should be applicable to a variety of diseases involving the hematopoietic stem cell.

本项目旨在开发白细胞粘附缺陷（LAD）作为一种 模型应用在干细胞生物学的理解进展， 造血干细胞的转导。LAD的特征在于 临床上通过复发性、危及生命的细菌感染， 白细胞不能粘附在血管壁上并迁移到血管壁， 感染部位。这些粘附缺陷源于 白细胞表达CD11/CD18白细胞 白细胞表面的整合素异源二聚体由于原发性遗传 CD18亚基的缺陷。 白细胞粘附缺陷是一个有吸引力的模型， 研究的几个原因：1）疾病是危及生命的， 严重缺乏的形式，除了骨髓移植有 是没有治疗以外的支持疗法; 2）疾病是由于一个 单个基因（CD18亚基）中的缺陷，并且该基因已被克隆; 3)已经显示正常CD 18基因转导到LAD EBV B细胞中 纠正生物化学和功能缺陷; 4）由骨引起 骨髓移植表明，缺陷存在于 造血干细胞; 5）基于白细胞的基因表达， LAD中度缺乏表型，CD 11/CD 18低水平 表达似乎足以纠正严重的临床 疾病的表现;以及6）由于缺陷涉及膜 受体，基因转移和表达的定量评估， 使用外周血白细胞的流式细胞术完成。 在当前的研究中，我们将在初步数据的基础上确定 逆转录病毒载体、包装细胞系和培养条件 促进长期培养起始细胞的转导。的 该项目的具体目标是：1）确定条件， 有效转导长期培养物所需的载体 启动细胞; 2）测定外周血干细胞的能力 将来自患有LAD的儿童的细胞用离体逆转录病毒转导， 介导的白细胞整合素CD 18亚单位的基因转导 随后再输注转导的细胞;和3）确定是否 预处理方案的预先给药增强了 白细胞整合素CD18亚基在离体后白细胞中的表达 CD 18转导入儿童外周血干细胞 小伙子 我们预计，这些研究将结合进展， 造血干细胞移植是造血干细胞移植的一个重要组成部分。 SCOR，包括那些识别诱导或选择茎的方法的SCOR， 最容易被转导的细胞， 支持造血细胞转导的载体和培养条件 干细胞这些研究的结果应该适用于各种 与造血干细胞有关的疾病