Lentiviral Gene Therapy of X-linked Agammaglobulinemia

描述(由申请人提供)：X连锁无丙种球蛋白血症(XLA)是由Bruton‘s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。对表达正常BTK的B细胞的选择性压力表明，将正常的BTK基因导入自体造血干细胞(HSC)可能会导致XLA的长期免疫重建。使用为表达小鼠HSC而优化的肿瘤逆转录病毒载体和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：完全挽救依赖BTK的原发和外周B细胞发育，纠正B细胞功能反应。这一数据表明，BTK基因转移可以在XLA的动物模型中重建BTK依赖的功能，并有力地支持这种治疗方法的进一步追求。目前的建议旨在解决推进最终XLA基因治疗所需的几个关键问题，包括开发慢病毒载体：将BTK基因高效地导入人HSC；将BTK基因特异性地靶向B系细胞；显著降低病毒增强子介导的病毒整合位点附近的突变风险。我们将测试假设：1)含有B系特异性启动子/增强子的慢病毒载体将在体内和体外介导持续的、暂时合适的BTK基因表达，并导致B细胞功能的挽救；以及2)在这些载体中加入绝缘体元件将促进提高表达，更重要的是，显著降低病毒增强子突变的风险。最近对原发免疫缺陷疾病的临床试验突出了遗传纠正的巨大潜力，以及发生肿瘤逆转录病毒相关恶性肿瘤的意外高风险。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，旨在开发安全和高效的载体系统，能够适当、特异和持续地表达B系基因。克服这些技术挑战也应该为缺乏选择性优势的先天性疾病的基因疗法的发展提供洞察力。

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.