Lentiviral Gene Therapy of X-Linked Agammaglobulinemia

描述（由申请方提供）：X连锁无丙种球蛋白血症（XLA）由布鲁顿酪氨酸激酶（Btk）功能缺陷引起，特征为早期B细胞发育严重阻滞。表达野生型Btk的B细胞在体内表现出很强的选择性优势;这表明将正常Btk cDNA引入自体造血干细胞（HSC）中可能导致XLA中的长期免疫重建。在以前的工作中，我们证明了γ-逆转录病毒和B谱系特异性的慢病毒（LV）Btk基因治疗可以导致Btk依赖性的拯救，B谱系的发展和功能在体内。值得注意的是，除了其在B细胞中的作用之外，Btk还通过在骨髓细胞上表达的多种受体被激活，这意味着Btk在宿主免疫应答中具有更广泛的作用。为了优化Btk转基因在B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）与两种替代元件[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来表达密码子优化的人Btk，我们观察到Btk依赖性B谱系发育和功能的拯救;以及骨髓细胞中Toll样受体（TLR）信号传导的恢复。总之，这些数据有力地支持了我们的目标，即在未来的XLA患者基因治疗试验中使用基于Btkp的LV。然而，在将这种方法应用于患者之前，仍有几个关键问题有待解决。首先，我们需要对EmBtkp与UCOE. BtkpLV在鼠XLA和来源于XLA患者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，我们需要详细评估这些候选LV的潜在遗传毒性。因此，该提议被设计为检验以下假设：（1）利用内源性Btk启动子的LV将介导持续的、时间上适当水平的Btk基因表达，并在鼠和人细胞中拯救B和骨髓功能;（2）使用非清髓性骨髓预处理方案也将实现有效的Btk拯救，该方案密切模拟XLA中的未来临床方法;和（3）这些LV将显示很少或没有载体介导的诱变的风险。

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.