GENE THERAPY IN PRIMARY IMMUNODEFICIENCY DISEASES

描述：（改编自申请人的摘要）：我们的最新进展 了解原发性免疫的分子和遗传基础 缺乏综合征使这些疾病成为基因治疗的主要候选者。 疗法 在大多数遗传决定的免疫缺陷中， 综合征，基因已被确定，突变引起的综合征 已经被发现，并且对免疫反应的分子后果 认可. 该项目的总体目标是开发技术， 纠正X连锁高IgM综合征（XHIM）的遗传缺陷， CD 40配体（CD 40 L）基因突变，并制定原则 基因治疗他们的原发性免疫缺陷疾病。 目前， 逆转录病毒载体提供了最有吸引力的方法， 将基因导入靶细胞，可以容易地培养，转导， 如本提案所述，可能会退还给捐助者。 我们有 构建了逆转录病毒载体LCD 40 LSN，其中人CD 40 L基因被 从病毒LTR表达，并高效转导该基因 未处理的不表达CD 40 L的正常人成纤维细胞。 在Aim中 1，我们建议生产一系列逆转录病毒载体， CD 40 L的组成型表达或活化诱导表达。 除了包含LTR的载体之外，我们将使用较弱的SV 40 用于组成型表达的启动子，以及人IL-2和CD 40 L启动子 活化诱导的CD 40 L表达。 在目标2中，我们将确定 T淋巴母细胞样细胞系高效转导的条件 来自XHIM患者，这些患者具有干扰 基因表达或导致非功能性蛋白质的表达。 的 将通过流式细胞术评估转导的T细胞系的CD 40 L表达。 流式细胞术和体外扁桃体B细胞培养物检测CD 40 L功能 系统 在目标3中，我们将使用在 小鼠CD 40 L基因转染骨髓干细胞的体外实验 CD 40配体敲除小鼠。 这将使我们能够测试在体内的影响， 表达的CD 40 L维斯活化诱导的 CD 40 L对受体小鼠免疫系统和临床表型的影响。 这些研究将为基因治疗铺平道路， 确定原发性免疫缺陷疾病。

DESCRIPTION: (Adapted from Applicant's Abstract): Recent advances in our understanding of the molecular and genetic basis of primary immune deficiency syndromes have made these disorders prime candidates for gene therapy. In the majority of the genetically determined immune deficiency syndromes, the gene has been identified, mutations causing the syndromes have been found, and the molecular consequences on the immune response recognized. The overall goal of this project is to develop techniques to correct the genetic defect in X-linked hyper IgM syndrome (XHIM), caused by the mutation of the CD40 ligand (CD40L) gene, and to work out the principles for gene therapy of their primary immunodeficiency disorders. Currently, retroviral vectors offer the most attractive method for the transfer of genes into target cells that can easily be cultured, transduced and, potentially, returned to the donor, as outlined in this proposal. We have constructed the retroviral vector, LCD40LSN in which the human CD40L gene is expressed from the viral LTR, and transduced the gene with high efficiency into normal human fibroblasts that untreated do not express CD40L. In Aim 1, we propose to produce a series of retroviral vectors that will result in either constitutive expression or activation-induced expression of CD40L. In addition to the LTR-containing vector, we will use the weaker SV40 promoter for constitutive expression, and the human IL-2 and CD40L promoters for activation-induced CD40L expression. In Aim 2, we will determine the conditions for high efficiency transduction of T lymphoblastoid cell lines derived from XHIM patients who have mutations that either interfere with gene expression or result in the expression of a nonfunctional protein. The transduced T cell lines will be assessed for CD40L expression by flow cytometry and for CD40L function by an in vitro tonsillar B cell culture system. In Aim 3, we will use the most suitable vectors identified by in vitro experiments to transfer murine CD40L gene in bone marrow stem cells of CD40 ligand knockout mice. This will allow us to test the in vivo effect of consituitively expressed CD40L vis-a-vis activation-induced expression of CD40L on the immune system and on the clinical phenotype of recipient mice. The studies will pave the way for gene therapy in patients will genetically determined primary immunodeficiency diseases.