GENE TRANSFER IN LEUKOCYTE ADHERENCE DEFICIENCY

这项提议的目的是使用逆转录病毒载体来引导 外周血中白细胞整合素CD18亚基的体外表达 正常人和正常人的血祖细胞和骨髓细胞 白细胞黏附缺陷或LAD的儿童，活体在骨中 CD18被基因打乱的小鼠骨髓细胞 瞄准目标。患有LAD的儿童反复发作，危及生命 细菌感染是由于这些细菌的白细胞不能 儿童附着在血管壁上并迁移到 感染。本病的临床表现源于 左前降支患儿外周血白细胞不能表达 细胞表面的白细胞整合素CD11/CD18分子 CD18亚单位缺陷。 白细胞黏附缺陷是人类基因的候选疾病 基于基因转移的造血祖细胞和 干细胞说：L)这种疾病在严重的情况下危及生命 缺乏型，2)除骨髓移植外 支持疗法以外的治疗，3)正常CD18的转导 基因进入LAD EBV B细胞已被证明能纠正生化和 功能缺陷，以及4)基于白细胞的基因表达。 LAD中度缺乏表型，CD11/CD18正常水平的5% 表达似乎足以纠正严重的临床 这种疾病的表现。 目前的研究旨在确定最优的条件 利用逆转录病毒载体恢复外周血中CD18的表达 LAD儿童的祖细胞和骨髓细胞的最终目标 CD18基因转入造血系统对疾病的长期纠正作用 干细胞。在这些研究中，我们的目标是：1)确定 逆转录病毒载体LgCD18SN和LgCD18所需的，编码人 CD18亚基，产生高效的基因转导和基因 外周血祖细胞CD18亚基的表达及意义 正常人和LAD患儿的骨髓细胞；2) 为了确定转导基因的疗效、效率和安全性。 人CD18亚基从小鼠骨髓细胞中转化为CD18 亚基已被基因打靶打乱。 LAD是一种很有吸引力人类基因治疗的模型 编码CD18的外周血祖细胞和骨髓细胞 载体在体外，然后重新输注转导细胞可能是 对患有LAD的儿童的主要治疗益处。在急性发作期间 感染粒细胞和单核细胞，区别于 CD18转导的祖细胞可以运输到炎症部位并 参与宿主对感染性病原体的反应。因此， 造血干细胞的转导可能不是必须的 LAD的临床反应。

The aim of this proposal is to use retroviral vectors to direct the expression of the leukocyte integrin CD18 subunit in vitro in peripheral blood progenitor and bone marrow cells from normal individuals and from children with leukocyte adherence deficiency or LAD, and in vivo in bone marrow cells from mice in which the CD18 has been disrupted by gene targeting. Children with LAD experience recurrent, life-threatening bacterial infections due to the inability of the leukocytes from these children to adhere to the vessel wall and migrate to the site of infection. The clinical manifestations of the disease stem from the inability of leukocytes from children with LAD to express the members of the leukocyte integrin CD11/CD18 molecules on the cell surface due to defects in the CD18 subunit. Leukocyte adherence deficiency is a candidate disease for human gene therapy based upon gene transfer into hematopoietic progenitor cells and stem cells in that: l) the disease is life-threatening in the severe deficiency form, 2) except for bone marrow transplantation there is no treatment other than supportive therapy, 3) transduction of a normal CD18 gene into LAD EBV B-cells has been shown to correct the biochemical and functional defect, and 4) based upon gene expression by leukocytes in the moderate deficiency phenotype of LAD, 5% of normal levels of CD11/CD18 expression appears to be sufficient to correct the severe clinical manifestations of the disease. The current studies are designed to determine the optimal conditions for using retroviral vectors to restore CD18 expression in peripheral blood progenitors and bone marrow cells from LAD children with the ultimate goal of long-term disease correction by CD18 gene transfer into hematopoietic stem cells. In these studies we aim to: 1) determine the conditions required for retroviral vectors LgCD18SN and LgCD18, encoding the human CD18 subunit, to produce high efficiency gene transduction and gene expression of the CD18 subunit in peripheral blood progenitor cells and bone marrow cells from normal individuals and from children with LAD; 2) to determine the efficacy, efficiency, and safety of transduction of the human CD18 subunit into bone marrow cells from mice in whom the CD18 subunit has been disrupted by gene targeting. LAD is an attractive model for human gene therapy in that transduction of peripheral blood progenitor cells and bone marrow cells with CD18-encoding vectors ex vivo followed by re-infusion of the transduced cells may be major therapeutic benefit to children with LAD. During acute episodes of infection the granulocytes and monocytes which differentiate from the CD18-transduced progenitor cells may traffic to the inflammatory site and participate in the host response to the infectious agent. Thus, transduction of a hematopoietic stem cell may not be required for a clinical response in LAD.