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: 7049380
• 负责人: David J Rawlings
• 金额: $ 30.65万
• 依托单位: SEATTLE CHILDREN'S HOSPITAL
• 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 2009-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7049380
• 关键词: Adenoviridae; B lymphocyte; NOD mouse; SCID mouse; Streptococcus pneumoniae; antibody formation; biological models; biological signal transduction; biotechnology; cell differentiation; clinical research; gene delivery system; gene expression; gene therapy; genetic promoter element; hematopoietic stem cells; human subject; hypogammaglobulinemia; immune response; patient oriented research; protein tyrosine kinase; septicemia; sex linked trait; stem cell transplantation; tissue /cell culture; transfection /expression vector

DESCRIPTION (provided by applicant): X-linked agammaglobulinemia (XLA) results from deficient function of Bruton's tyrosine kinase (Btk) and is characterized by a severe block in early B-cell development. The selective pressure for B cells expressing normal Btk suggests that introduction of the Btk cDNA into autologous hematopoietic stem cells (HSC) may lead to long-term immunologic reconstitution in XLA. Using retroviral vectors optimized for expression in HSC and transplantation of transduced stem cells into Btk/Tec-/- recipients we have achieved: sustained marking and B lineage engraftment, and selective expansion of Btk expressing B lineage cells. This has led to full restoration of both the pre-B and transitional B cell development transitions, and of Bkt dependent functions including B cell receptor signaling, T-independent immune responses, and circulating immunoglobulin. In the current proposal we will refine this viral delivery system to optimize its clinical feasibility for rescue of Btk function. We will test the hypotheses that: 1.) The selective outgrowth of the corrected cells will be sufficient to restore Btk dependent functions in a minimally or non-myeloablated setting; 2) The rescue of B cell development and function will protect gene corrected animals from life-threatening bacteremia; 3) Self-inactivating retroviral vectors containing unique B-lineage enhancer/promoter elements will mediate sustained, temporally appropriate Btk expression and rescue function in vivo and in vitro; 4) Incorporation of the insulator elements into these SIN vectors will promote more consistent expression and, more importantly, reduce the risk of viral enhancer mediated mutagenesis by limiting activation of host gene expression; 5) These viral vectors will rescue the deficient generation of surface lg+ B cells from transduced XLA HSC in vitro and in vivo. Recent clinical trials in immunodeficiency disorders highlight the great potential for genetic correction as well as an unanticipated risk for development of onco-retroviral toxicity. While conceptually simple, implementation of definitive genetic therapy in XLA requires a concerted program of basic and applied research aimed at developing safe and efficient vector systems capable of appropriate, specific and sustained B lineage gene expression. Overcoming these technical challenges will likely provide important insight into therapies for other more common genetic disorders lacking a selective advantage for corrected cells.

描述（由申请人提供）：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，其特征是早期B细胞发育严重受阻。表达正常 Btk 的 B 细胞的选择压力表明，将 Btk cDNA 引入自体造血干细胞 (HSC) 可能会导致 XLA 的长期免疫重建。使用针对 HSC 中表达进行优化的逆转录病毒载体并将转导干细胞移植到 Btk/Tec-/- 受体中，我们实现了：持续标记和 B 谱系植入，以及表达 Btk 的 B 谱系细胞的选择性扩增。这导致前 B 细胞和过渡 B 细胞发育转变以及 Bkt 依赖性功能（包括 B 细胞受体信号传导、T 依赖性免疫反应和循环免疫球蛋白）的完全恢复。在当前的提案中，我们将完善该病毒递送系统，以优化其挽救 Btk 功能的临床可行性。我们将测试以下假设： 1.) 校正细胞的选择性生长将足以在最低限度或非清髓环境中恢复 Btk 依赖性功能； 2）拯救B细胞的发育和功能将保护基因纠正的动物免受危及生命的菌血症； 3) 含有独特B谱系增强子/启动子元件的自失活逆转录病毒载体将在体内和体外介导持续的、暂时适当的Btk表达和救援功能； 4) 将绝缘体元件掺入这些SIN载体中将促进更一致的表达，更重要的是，通过限制宿主基因表达的激活来降低病毒增强子介导的诱变的风险； 5) 这些病毒载体将在体外和体内挽救转导的 XLA HSC 表面 lg+ B 细胞的缺陷生成。最近的免疫缺陷疾病临床试验强调了基因校正的巨大潜力以及发生肿瘤逆转录病毒毒性的意外风险。虽然概念上很简单，但在 XLA 中实施明确的基因治疗需要协调一致的基础和应用研究计划，旨在开发能够适当、特异性和持续 B 谱系基因表达的安全有效的载体系统。克服这些技术挑战可能会为其他更常见的遗传性疾病的治疗提供重要的见解，这些遗传性疾病缺乏校正细胞的选择性优势。