MODEL FOR GENE THERAPY IN X LINKED AGAMMAGLOBULINEMIA

X连锁无丙种球蛋白血症的基因治疗模型

• 批准号: 2829859
• 负责人: David J Rawlings
• 金额: $ 18.06万
• 依托单位: UNIVERSITY OF CALIFORNIA LOS ANGELES
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-06-01 至 2004-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2829859
• 关键词: Adenoviridae; B lymphocyte; Epstein Barr virus; NOD mouse; SCID mouse; antibody formation; biological models; biological signal transduction; calcium flux; cell differentiation; gene therapy; hematopoietic stem cells; hypogammaglobulinemia; model design /development; recombinant virus; sex linked trait; tissue /cell culture; transfection /expression vector

DESCRIPTION: (Adapted from applicant's abstract). Human X-linked agammaglobulinemia (XLA) results from deficient function of Bruton's tyrosine kinase (Btk). In affected males, the production of B lineage cells is profoundly reduced resulting in life threatening humoral immunodeficiency. Definitive genetic therapy utilizing expression of wild type Btk in stem cells or B lineage progenitors would represent a significant advantage over the current supportive management of XLA. The Investigator proposes to establish pre-clinical cellular models for the genetic treatment of XLA through three aims. Aim 1. Development of a multistage human B cell culture system that can be used to evaluate reconstitution of B cell function in vitro. The Investigator will improve the derivation of B cells representing multiple stages of the B lineage from long-term human B progenitor cell cultures. This will be done by exploiting a range of culture modifications and signals that lead to activation, maturation, and production of secreted immunoglobulin by immature B cells. Soluble factors and cell surface ligands on stromal cells will be evaluated. The Investigator will also utilize in vivo transfer studies in SCID/NOD mice. Aim 2: Development and testing of the capacity of retroviral vectors capable of high level, sustained expression in hematopoietic stem cells, and of novel hybrid adenoviral/EBV episome vectors for Btk gene transfer and rescue of Btk signaling in vitro. Alternative gene transfer systems will be evaluated using a new signaling competent human B cell model developed by the Investigator's laboratory. This will allow the Investigator to rapidly test the transduction efficiency, and the levels and duration of protein expression of alternative vectors. In addition, the Investigator will directly evaluate the capacity of these vectors to rescue Btk dependent signaling events and determine the dosage of Btk required for functional reconstitution in human B cells. Aim 3. Evaluation of the capacity of those gene transfer vectors that successfully restore Btk signaling in vitro to reconstitute Btk dependent immune function in vivo in Btk-/- mice, and ultimately, in vitro in long term cultured XLA B cells. The Investigator will evaluate gene transfer using hematopoietic stem cells and growth factor expanded B progenitors from Btk-/-mice as target cell populations. Reconstitution of B lineage development and function will be evaluated using multiple measures of Btk dependent immune function. Those vectors demonstrating optimal restoration of Btk function in vivo will be utilized for gene transfer into stem cells or B progenitors in pre-clinical studies of patients with XLA.

描述：（改编自申请人的摘要）。人类 X 连锁无丙种球蛋白血症 (XLA) 是由布鲁顿酪氨酸激酶 (Btk) 功能缺陷引起的。在受影响的男性中，B 谱系细胞的产生大大减少，导致危及生命的体液免疫缺陷。利用干细胞或 B 谱系祖细胞中野生型 Btk 表达的确定性基因治疗将比目前的 XLA 支持性治疗具有显着优势。研究者建议通过三个目标建立 XLA 基因治疗的临床前细胞模型。目标 1. 开发多阶段人类 B 细胞培养系统，可用于评估体外 B 细胞功能的重建。研究者将改进从长期人类 B 祖细胞培养物中代表 B 谱系多个阶段的 B 细胞的衍生。这将通过利用一系列培养物修饰和信号来实现，这些修饰和信号会导致未成熟 B 细胞激活、成熟和产生分泌的免疫球蛋白。将评估基质细胞上的可溶性因子和细胞表面配体。研究人员还将利用 SCID/NOD 小鼠的体内转移研究。目标 2：开发和测试能够在造血干细胞中高水平、持续表达的逆转录病毒载体的能力，以及用于 Btk 基因转移和体外 Btk 信号转导的新型混合腺病毒/EBV 附加体载体的能力。将使用研究者实验室开发的新的具有信号传导能力的人类 B 细胞模型来评估替代基因转移系统。这将使研究人员能够快速测试替代载体的转导效率以及蛋白质表达的水平和持续时间。此外，研究者将直接评估这些载体拯救 Btk 依赖性信号传导事件的能力，并确定人类 B 细胞功能重建所需的 Btk 剂量。目标 3. 评估那些在体外成功恢复 Btk 信号传导的基因转移载体的能力，以在 Btk-/- 小鼠体内重建 Btk 依赖性免疫功能，并最终在体外长期培养的 XLA B 细胞中重建 Btk 依赖性免疫功能。研究者将使用来自 Btk-/-小鼠的造血干细胞和生长因子扩增的 B 祖细胞作为靶细胞群来评估基因转移。将使用 Btk 依赖性免疫功能的多种测量来评估 B 谱系发育和功能的重建。这些在体内表现出 Btk 功能最佳恢复的载体将用于在 XLA 患者的临床前研究中将基因转移到干细胞或 B 祖细胞中。