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.

该项目旨在发展白细胞黏附缺陷(LAD)作为一种 将干细胞生物学的研究进展应用于 造血干细胞的转导。LAD的特征是 临床上由反复发生的危及生命的细菌感染引起 白细胞不能附着在血管壁上并迁移到 感染部位。这些粘着缺陷源于不能 患儿外周血白细胞表达CD11/CD18的研究 白细胞表面整合素异二聚体的原发遗传 CD18亚单位缺陷。 白细胞黏附缺陷是一种有吸引力的模型 研究有几个原因：1)这种疾病在中国是危及生命的 严重缺乏型，除骨髓移植外 除了支持性治疗外没有其他治疗方法；2)这种疾病是由于 缺陷单基因CD18亚基，该基因已被克隆； 3)将正常的CD18基因导入LAD EBV B细胞 纠正生化和功能缺陷；4)由骨骼引起 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表型中度缺乏，CD11/CD18水平低 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，可以定量评估基因的转移和表达 采用流式细胞术检测外周血白细胞。 在目前的研究中，我们将以初步数据为基础，确定 逆转录病毒载体、包装细胞系和培养条件 促进长期培养启动细胞的转导。这个 本项目的具体目标是：1)确定条件和 有效转导长期培养所需的载体 2)外周血干/祖细胞能力测定 来自LAD儿童的细胞将通过体外逆转录病毒转导- 白细胞整合素CD18亚单位介导的基因转导 然后重新输注被转导的细胞；以及3)确定 预先给予一种条件化疗法可增强 白细胞整合素CD18亚基在体外培养中的变化 CD18转导儿童外周血干细胞的实验研究 小伙子。 我们预计这些研究将纳入以下方面的进展 从中国的其他项目中了解造血干细胞 SCOR，包括那些识别诱导或选择茎的方法 最容易转导的细胞，以及那些正在发育的细胞 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不同的 涉及到造血干细胞的疾病。