HBF Variants for Gene Therapy of Sickle Cell Disease

描述（由申请人提供）：尽管对Hb进行了广泛的研究， 血红素和珠蛋白亚基协同组装的机制 以及错误折叠和不稳定的未组装球蛋白链是如何从 红细胞未知。此外，Hb F 抑制聚合和改善SCD的临床过程， 完全理解阐明这种机制有助于 发展基因治疗的策略， 改变的珠蛋白链或与珠蛋白合成减少相关的那些。在 本提案的目的是（1）γ-链组装与α-链形成功能性 人胎儿Hb，（2）泛素介导过量非α珠蛋白降解 （3）具有低氧亲和力的工程化Hb F变体， 抑制Rb S聚合。长期目标是 鉴定和设计用于镰刀基因治疗最佳Rb F变体 细胞病（SCD）和地中海贫血。在具体目标（1）中，我们将测试两个 相关假设;（i）折叠的α-珠蛋白链与中间组装 折叠的新生γ-链之前或之后不久释放， 多核糖体(ii)G-10、14和18处的氨基酸，其已经显示 通过X射线晶体学分析，在G上的α 1 γ 1相互作用位点上 螺旋，对于体内α-和γ-珠蛋白链组装以及 体外在特异性目的（2）中，我们假设纯化的非α链 四聚体，如血红蛋白异源四聚体，不是泛素化的底物 由于β 4和γ 4结构与α 2 β 2异源四聚体非常相似 结构使用兔网织红细胞无细胞系统，我们将测量 在不存在链的情况下，非α链在翻译过程中的降解 泛素的存在。在具体目标（3）中，我们将继续调查 Hb F抑制Hb S聚合机制我们假设Hb F 变体（例如，Hb F γ 73 His、Rb F γ 6Val和73 His）可以被工程化， 抑制特性超过Hb F的那些，并且我们将寻找这样的变体。 我们还将继续寻找氧亲和力低于Hb的Rb F变体 S不仅通过增强2，3-BPG相互作用， 在G螺旋上的酰基相互作用位点处的取代。因为他们的 较低的氧亲和力，这些血红蛋白变体除了具有 反成核特性将有效地抑制在较低水平的镰状化 比天然Rb F更高，例如约10%对20%。的理解 γ和α链的组装以及过量的 珠蛋白将为确定最合适的γ链突变体提供基础 用于基因治疗，这应该是一种可以通过病毒载体引入的基因治疗， 显著低于天然Rb F。此外，这些研究将 研究蛋白质生物合成的研究人员普遍感兴趣， 有助于确定为什么一些突变的珠蛋白链被纳入血红蛋白 比野生型链效率更高或更低， 在血红蛋白形成过程中对翻译α和非α链进行质量控制 以保存功能性红细胞。

DESCRIPTION (provided by applicant): Despite extensive research on the Hb molecule, the mechanism by which heme and globin subunits coordinately assemble and how misfolded and unstable unassembled globin chains are removed from erythrocytes are not known. In addition, the basic mechanism by which Hb F inhibits polymerization and ameliorates the clinical course of SCD is not completely understood. Elucidating such mechanisms can contribute to the development of strategies for gene therapy in the treatment of diseases of altered globin chains or those associated with decreased globin synthesis. In this proposal we aim (1) gamma-chain assembly with a chains to form functional human fetal Hb, (2) Ubiquitin-mediated degradation of excess non-alpha globin chains in vivo, and (3) Engineered Hb F variants having low oxygen affinity and inhibitory properties on Rb S polymerization. The long-range goal is to identify and design optimal Rb F variants for use in gene therapy of sickle cell disease (SCD) andthalassemia. In Specific Aims (1) we will test two related hypotheses; (i) Folded alpha-globin chains assemble with intermediately folded nascent gamma-chains prior to or soon after the release from polyribosomes. (ii) The amino acids at G-10, 14 and 18, which have been shown by x-ray crystallographic analysis to be at the alpha1gamma1 interaction sites on the G helix, are critical for assembly of alpha- and gamma-globin chains in vivo as well as in vitro. In Specific Aim (2), we hypothesize that purified non-alpha chain tetramers, like Hb hetero-tetramers, are not substrates for ubiquitination since Beta4 and gamma4 structures are very similar to the alpha2Beta2 heterotetramer structure. Using a rabbit reticulocyte cell free system, we will measure degradation of non-alpha chain in the absence of a chain during translation in the presence of ubiquitin. In specific Aim (3), we will continue to investigate the inhibitory mechanism of Hb S polymerization by Hb F. We hypothesize that Hb F variants (e.g., Hb F gamma 73 His, Rb F gamma 6Val & 73 His) can be engineered that have inhibitory properties exceeding those of Hb F and we will seek such variants. We will also continue to seek Rb F variants with lower oxygen affinity than Hb S through not only enhancement of 2,3-BPG interaction but also amino acid substitution at the alyl interaction sites on the G helix. Because of their lower oxygen affinity, these hemoglobin variants in addition to having anti-nucleation properties would effectively inhibit sickling at lower levels than would native Rb F, such as about 10 percent vs. 20 percent. The understanding of the assembly of gamma and alpha chain and the mechanism of degradation of excess globin will provide a basis for determining the most appropriate gamma chain mutant for gene therapy, which should be one that can be introduced by viral vectors at significantly lower levels than native Rb F. Furthermore, these studies will be of general interest to researchers who study protein biosynthesis and will help identify why some mutant globin chains are incorporated into hemoglobin more or less efficiently than wild type chains as well as how separately translated alpha and non-alpha chain are quality controlled during hemoglobin formation to preserve functional erythrocytes.