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。表达野生型Btk的B细胞在体内表现出很强的选择优势；这表明将正常的Btk cDNA引入自体造血干细胞（HSC）可能导致XLA的长期免疫重建。在之前的工作中，我们证明了γ -逆转录病毒和B谱系特异性慢病毒（LV） Btk基因治疗都可以在体内挽救Btk依赖的B谱系发育和功能。值得注意的是，除了在B细胞中发挥作用外，Btk还通过髓细胞上表达的多个受体被激活，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系和髓系细胞中的表达谱，我们开发了一个新的LV平台，使用内源性Btk启动子（Btkp）与两个替代元件（IgH链增强子Em或普遍存在的染色质开放元件（UCOE）元件）相关联。利用这些LV表达经过密码子优化的人Btk，我们观察到Btk依赖性的恢复，b谱系的发育和功能；和髓细胞toll样受体（TLR）信号的恢复。总之，这些数据有力地支持了我们在未来XLA患者基因治疗试验中使用基于btkp的LV的目标。几个关键问题；然而，在将这种方法推向患者之前，仍有待解决。首先，我们需要对EmBtkp与UCOE的安全性和有效性进行全面的体内比较。小鼠XLA和来自XLA患者的原代干细胞中的Btkp LV。其次，我们需要详细评估这些候选LV的潜在遗传毒性。因此，本提案旨在验证以下假设：(1)利用内源性Btk启动子的LV将介导小鼠和人类细胞中持续、暂时适当水平的Btk基因表达和B和髓系功能的恢复；(2)使用非清髓性骨髓调节方案也可以实现有效的Btk拯救，该方案与XLA的未来临床方法非常相似；(3)这些lv将表现出很少或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.