Development of gene therapy for wiskott-aldrich syndrome (WAS)

威斯科特-奥尔德里奇综合征 (WAS) 基因疗法的开发

• 批准号: 7784215
• 负责人: ARTHUR W. NIENHUIS
• 金额: $ 35.27万
• 依托单位: ST. JUDE CHILDREN'S RESEARCH HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-09-01 至 2015-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7784215
• 关键词: Advanced Development; Affect; Allogenic; Autoimmunity; Autologous; Biological Assay; Blood; Blood Cells; Bone Marrow Cells; Bone Marrow Transplantation; Busulfan; CD34 gene; CSF3 gene; Cell Line; Cells; Cellular Assay; Chromatin; Chromatin Structure; Chronic; Clinical; Clinical Trials; Code; Data; Development; Disease; Distant; Eczema; Elements; Enhancers; Event; Future; Gene Expression; Gene Expression Regulation; Gene Proteins; Gene Transduction Agent; Gene Transfer; Genes; Goals; HIV-1; Hematopoietic; Human; Immunoglobulin M; Immunologic Deficiency Syndromes; Infusion procedures; Insulator Elements; Introns; Lentivirus Vector; Long Terminal Repeats; Lymphoid; Lymphoid Cell; Maps; Measures; Mediating; Methodology; Modality; Monitor; Mus; Myelogenous; Myeloid Cells; Myelosuppression; Myelosuppressive Therapy; Neoplasms; Oncogene Activation; Oncogenes; Participant; Patients; Peripheral Blood Stem Cell; Phase; Plasmid Cloning Vector; Platelet Count measurement; Preparation; Proteins; Proto-Oncogenes; Protocols documentation; Reading; Regulatory Element; Relative Risks; Research; Resolution; Safety; Site; Stem cell transplant; Stem cells; System; Technology; Testing; Therapeutic; Thrombocytopenia; Tissues; Vesicular stomatitis Indiana virus; Wiskott-Aldrich Syndrome; Work; X-Linked Severe Combined Immunodeficiency; base; boys; cell immortalization; cellular transduction; design; eligible participant; env Gene Products; gene therapy; genetically modified cells; immune function; in vivo; insight; internal control; leukemia; meetings; novel; promoter; protein expression; reconstitution; research study; restoration; vector; vector genome; vector-induced

This project is focused on the development of gene therapy for Wiskott-Aldrich syndrome (WAS), a severe immunodeficiency disorder also characterized by a low platelet count and chronic eczema. Affected boys may also suffer from autoimmunity and/or develop a neoplasm. We have made the following advances which support the application of gene transfer into blood stem cells for treatment of WAS: 1) developed a novel, HIV1 based lentiviral vector system to facilitate gene transfer into stem cells; 2) identified the envelope protein from Vesicular Stomatitis Virus (VSV-G) as providing the highest efficiency of gene transfer into repopulating cells; and 3) developed a methodology for deriving stable producer clones that will facilitate vector preparation for our planned clinical trial. In Specific Aim 1, experiments are proposed to identify a lentiviral vector design that achieves normal expression of Wiskott-Aldrich syndrome protein (WASp) in hematopoietic cells and demonstrates therapeutic potential, in Sub-Aim 1.1, we will compare the levels of WASp expression achieved with various promoters to select one for use in our clinical trial. In a second exploratory sub-aim of Specific Aim 1, we will map and functionally characterize distant tissue specific regulatory elements that may influence WASp gene expression. In Specific Aim 2, we will evaluate the safety of WASp clinical vector using 2 cellular assays that detect proto-oncogene activation. Vectors will be assayed for their potential to activate the LM02 proto-oncogene in Jurkat T-celis and also for their ability to induce myeloid immortalization of primary lineage depleted bone marrow cells. In Specific Aim 3, we propose to evaluate lentiviral vector mediated WASp gene transfer in WAS patients. Clinical vector design will be determined by the functional studies proposed in Sub-Aim 1.1 as well as the cellular assays for protooncogene activation in Specific Aim 2. Eligible participants are those whose platelet count is < 50,000/mm3 who have other significant clinical manifestations of WAS but lack a matched related or unrelated allogeneic stem cell donor. G-CSF mobilized peripheral blood stem cells will be transduced and returned to participants following myelosuppressive therapy with Busulfan. Safety and feasibility will be assessed within 2 months of infusion of transduced cells and the protocol amended if one or more stopping rules are met. Objective measures of efficacy include a progressive increase in the number of genetically modified cells, particulariy in the lymphoid lineages, an increase in platelet count to &50,000/mm3 and a return of IgM levels to normal by 1 year. Patients will be observed long-term for restoration of immune function and for any evidence of vector induced clonal dominance or neoplasia.

该项目的重点是开发Wiskott-Aldrich综合征（WAS）的基因治疗，WAS是一种严重的免疫缺陷疾病，其特征还包括血小板计数低和慢性湿疹。受影响的男孩也可能患有自身免疫和/或发展肿瘤。我们已经取得了以下进展，支持将基因转移到血液干细胞中用于治疗WAS的应用：1）开发了一种新的基于HIV 1的慢病毒载体系统，以促进基因转移到干细胞中; 2）鉴定了来自水泡性口炎病毒（VSV-G）的包膜蛋白，其提供了最高效率的基因转移到再生细胞中;和3）开发了用于获得稳定的生产克隆的方法，其将有助于我们计划的临床试验的载体制备。在具体目标1中，提出了实验来确定 在子目标1.1中，我们将比较用各种启动子实现的WASp表达水平，以选择一种用于我们的临床试验。在具体目标1的第二个探索性子目标中，我们将绘制可能影响WASp基因表达的远距离组织特异性调控元件并对其进行功能表征。在特定目标2中，我们将使用2种检测原癌基因激活的细胞试验评价WASp临床载体的安全性。将测定载体在Jurkat T细胞中激活LM 02原癌基因的潜力以及诱导原代谱系耗尽的骨髓细胞的髓样永生化的能力。在具体目标3中，我们建议 评价慢病毒载体介导的WASp基因转移在WAS患者中的作用。临床载体设计将通过子目标1.1中提出的功能研究以及特定目标2中原癌基因激活的细胞试验来确定。符合条件的参与者是血小板计数<50，000/mm 3且具有WAS的其他显著临床表现但缺乏匹配的相关或不相关异基因干细胞供体的那些人。G-CSF动员的外周血干细胞将被转导并在白消安骨髓抑制治疗后返回给受试者。将在输注转导细胞后2个月内评估安全性和可行性，如果符合一个或多个停止规则，则修改方案。目的 疗效的测量包括遗传修饰细胞数量的逐渐增加，特别是在淋巴谱系中，血小板计数增加到< 50，000/mm 3，IgM水平在1年时恢复到正常。将长期观察患者的免疫功能恢复情况以及任何载体诱导的克隆优势或肿瘤形成的证据。