GENE THERAPY IN PRIMARY IMMUNODEFICIENCY DISEASES

描述：(改编自申请人摘要)：我们的最新进展 了解初级免疫的分子和遗传学基础 虚证使这些疾病成为基因的主要候选者 心理治疗。在大多数遗传决定的免疫缺陷中 综合征，基因已经确定，突变导致了综合征 已经发现，免疫反应的分子后果 被认可了。该项目的总体目标是开发技术来 纠正X-连锁高IgM综合征(XHIM)的遗传缺陷，由 CD40配体(CD40L)基因的突变及其原理 用于对他们的原发免疫缺陷疾病进行基因治疗。目前， 逆转录病毒载体提供了最有吸引力的转移方法 基因进入靶细胞，可以很容易地培养、转导和， 如本提案所述，有可能退还给捐赠者。我们有 构建人CD40L基因逆转录病毒载体LCD40LSN 从病毒LTR中表达，并高效转导该基因 转化为正常的人成纤维细胞，未经处理不表达CD40L。在AIM 1，我们建议生产一系列逆转录病毒载体，将导致 CD40L的组成性表达或激活诱导表达。 除了包含Ltr的载体外，我们还将使用较弱的SV40 组成型表达启动子及人IL-2和CD40L启动子 用于激活诱导的CD40L表达。在目标2中，我们将确定 T淋巴母细胞系高效转导条件的研究 来自XHIM患者，他们的突变要么干扰 基因表达或导致非功能蛋白质的表达。这个 转导的T细胞株将通过Flow评估CD40L的表达 细胞计数法检测扁桃体B细胞CD40L功能 系统。在目标3中，我们将使用由中确定的最合适的载体 小鼠CD40L基因在小鼠骨髓干细胞中转移的体外实验 CD40配体基因敲除小鼠。这将使我们能够测试在体内的效果 CD40L与活化诱导表达CD40L CD40L对免疫系统和受体小鼠临床表型的影响。 这些研究将为基因治疗患者的基因治疗铺平道路 确诊的原发免疫缺陷疾病。

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.