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。表达野生型BTK的B细胞在体内表现出很强的选择性优势；提示将正常的BTK基因导入自体造血干细胞(HSC)可能导致XLA的长期免疫重建。在以前的工作中，我们证明了伽玛逆转录病毒和B系特异性慢病毒(LV)BTK基因治疗都可以挽救BTK依赖的B系在体内的发育和功能。值得注意的是，除了在B细胞中的作用外，BTK还通过髓系细胞上表达的多种受体被激活，这意味着BTK在宿主免疫反应中发挥了更广泛的作用。为了优化BTK转基因在B细胞和髓系细胞中的表达谱，我们开发了一种新的LV平台，该平台使用内源性BTK启动子(Btkp)和两个可选元件[IgH链增强子Em或普遍存在的染色质开放元件(UCOE)元件]。使用这些LV表达密码子优化的人BTK，我们观察到BTK依赖的B系发育和功能的挽救，以及髓系细胞中Toll样受体(TLR)信号的恢复。总之，这些数据有力地支持了我们的目标，即在未来针对XLA患者的基因治疗试验中使用基于Btkp的LV。然而，在这种方法应用于患者之前，仍有几个关键问题需要解决。首先，我们需要对EmBtkp和UCOE.Btkp LV在小鼠XLA和XLA患者来源的原代干细胞中的安全性和有效性进行全面的体内比较。其次，我们需要对这些候选LV的潜在遗传毒性进行详细评估。因此，这项建议旨在检验如下假设：(1)利用内源性BTK启动子的LV将在小鼠和人类细胞中介导持续、暂时适当的BTK基因表达水平以及B和B的髓系功能的挽救；(2)BTK的有效挽救也将使用紧密模仿XLA未来临床方法的非清髓性骨髓调节方案来实现；(3)这些LV很少或根本没有载体介导的突变风险。

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. B cells that express wildtype Btk exhibit a strong selective advantage in vivo; suggesting that introduction of a normal Btk cDNA into autologous hematopoietic stem cells (HSC) may lead to long-term immunologic reconstitution in XLA. In previous work, we demonstrated that both gamma-retroviral and B lineage-specific, lentiviral (LV) Btk gene therapy can lead to rescue of Btk-dependent, B lineage development and function in vivo. Notably, in addition to its role in B cells, Btk is activated via multiple receptors expressed on myeloid cells implying a broader role for Btk in host immune responses. To optimize the expression profile of the Btk transgene in both B and myeloid lineage cells, we developed a novel LV platform using the endogenous Btk promoter (Btkp) in association with two alternative elements [the IgH chain enhancer Em or a ubiquitous chromatin opening element (UCOE) element]. Using these LV to express codon-optimized human Btk, we observed rescue of Btk-dependent, B-lineage development and function; and restoration of Toll-like receptor (TLR) signaling in myeloid cells. Together, these data strongly support our goal of using a Btkp-based LV in a future gene therapy trial for patients with XLA. Several key issues; however, remain to be addressed before this approach can be moved forward into patients. First, we need a comprehensive in vivo comparison of the safety and efficacy of EmBtkp vs. UCOE.Btkp LV in both murine XLA and primary stem cells derived from XLA patients. Second, we need a detailed assessment of potential genotoxicity of these candidate LV. Accordingly, this proposal is designed to test the hypotheses that: (1) LV utilizing the endogenous Btk promoter will mediate sustained, temporally appropriate levels of Btk gene expression and rescue of B and myeloid function in murine and human cells; (2) Efficient Btk rescue will also be achieved using a non-myeloablative marrow conditioning regimen that closely mimics a future clinical approach in XLA; and (3)These LVs will exhibit little or no risk of vector- mediated mutagenesis.