Lentiviral Gene Therapy of X-linked Agammaglobulinemia

描述（由申请人提供）：X-linked agammaglobulinemia （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细胞发育严重阻滞。表达正常Btk的B细胞的选择压力表明，将正常Btk cDNA引入自体造血干细胞（HSC）可能导致XLA的长期免疫重建。利用优化的逆转录病毒载体表达小鼠HSC，并将转导的干细胞移植到Btk/Tec双缺陷受体中，我们最近实现了：完全恢复原代和外周Btk依赖的B细胞发育，并纠正B细胞功能反应。这些数据表明，Btk基因转移可以在XLA动物模型中重建Btk依赖的功能，并有力地支持了这种治疗方法的进一步研究。目前的提案旨在解决推进最终XLA基因疗法开发所需的几个关键问题，包括慢病毒载体的开发：介导高效Btk基因传递到人HSC；特异性靶向B系细胞表达Btk；并且在病毒整合位点附近表现出显著降低的病毒增强子介导的突变风险。我们将验证以下假设：1)含有B谱系特异性启动子/增强子的慢病毒载体将介导持续的、暂时适当水平的Btk基因表达，并导致体内和体外B细胞功能的恢复；2)在这些载体中加入绝缘体元件将促进表达的改善，更重要的是，显著降低病毒增强子突变的风险。最近针对原发性免疫缺陷疾病的临床试验强调了基因校正的巨大潜力，以及意想不到的与逆转录病毒相关的恶性肿瘤发展的高风险。虽然概念上很简单，但在XLA中实施明确的基因治疗将需要一个协调一致的基础和应用研究计划，旨在开发安全有效的载体系统，能够适当、特异性和持续地表达B谱系基因。克服这些技术挑战也应该为缺乏选择优势的先天性疾病的基因治疗的发展提供见解。

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 a normal Btk cDNA into autologous hematopoietic stem cells (HSC) may lead to long-term immunologic reconstitution in XLA. Using onco-retroviral vectors optimized for murine HSC expression and transplantation of transduced stem cells into Btk/Tec doubly deficient recipients we have recently achieved: full rescue of both primary and peripheral Btk-dependent B-cell development, and correction of B cell functional responses. This data demonstrates that Btk gene transfer can reconstitute Btk-dependent functions in an animal model of XLA, and strongly support the further pursuit of this therapeutic approach. The current proposal seeks to address several key issues required to move forward with development of a definitive XLA genetic therapy including development of lentiviral vectors that: mediate highly efficient Btk gene delivery into human HSC; specifically target Btk expression to B lineage cells; and exhibit a significantly reduced risk of viral enhancer mediated mutagenesis proximal to the sited of viral integration. We will test the hypotheses that: 1) Lentiviral vectors containing B-lineage specific promoter/enhancers will mediate sustained, temporally appropriate levels Btk gene expression and lead to rescue of B cell function in vivo and in vitro; and that 2) Incorporation of an insulator elements into these vectors will promote both improved expression and, more importantly, a significantly reduced risk for of viral enhancer mutagenesis. Recent clinical trials for primary immunodeficiency disorders highlight the great potential for genetic correction as well as an unanticipated, high risk for development of onco-retroviral associated malignancies. While conceptually simple, implementation of definitive genetic therapy in XLA will require 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 should also provide insight for development of gene therapy in congenital diseases that lack a selective advantage.