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: 6202424
• 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: 1999
• 资助国家: 美国
• 起止时间: 1999-09-01 至 2000-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6202424
• 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.

该项目旨在将白细胞依从性缺乏（LAD）作为一个 应用于理解干细胞生物学的进步的模型 造血干细胞的转导。小伙子是特征的 在临床上，由于复发性，威胁生命，细菌感染而引起的 白细胞无法粘附在容器壁上并迁移到 感染部位。这些依从性缺陷源于无法 来自受影响儿童的白细胞表达CD11/CD18白细胞 由于原发性遗传 CD18亚基中的缺陷。 白细胞依从性缺乏是提议的有吸引力的模型 研究有几个原因：1）该疾病正在威胁生命 严重的缺乏形式，除了那里的骨髓移植 除了支持治疗外，没有其他治疗； 2）疾病是由于 单个基因的缺陷，CD18亚基和基因已克隆。 3）已显示了正常CD 18基因到LAD EBV B细胞中的转导 纠正生化和功能缺陷； 4）骨骼的结果 骨髓移植表明缺陷位于 造血干细胞； 5）基于白细胞中的基因表达 LAD的中等缺陷表型，低水平的CD11/CD18 表达似乎足以纠正严重的临床 疾病的表现； 6）由于缺陷涉及膜 受体，基因转移和表达的定量评估可以是 使用外周血白细胞的流式细胞仪完成。 在当前的研究中，我们将基于我们的初步数据识别 逆转录病毒载体，包装细胞系和培养条件 促进长期培养引发细胞的转导。这 该项目的具体目的是：1）确定条件和 有效转导长期培养所需的向量 引发细胞； 2）确定外周血茎的能力 来自LAD儿童的细胞使用离体逆转录病毒转导的细胞 白细胞整合素CD 18亚基的介导的基因转导 然后再灌注转导的细胞。 ，3）确定是否 调节方案的事先给药增强了 白细胞整联蛋白CD18亚基在ex vivo之后 将CD 18转导到来自患有儿童的外周血干细胞 小伙子。 我们预计这些研究将纳入 了解其他项目的造血干细胞 SCOR，包括那些识别引起或选择茎的方式 最容易被转导的细胞，以及开发的细胞 载体和培养条件支持造血的转导 干细胞。这些研究的结果应适用于多种 涉及造血干细胞的疾病。