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 分子，血红素和珠蛋白亚基协调组装的机制 以及错误折叠和不稳定的未组装的珠蛋白链是如何从 红血球是未知的。此外，Hb F的基本机制 抑制聚合和改善SCD的临床病程 完全理解。阐明这些机制有助于 基因治疗在疾病治疗中的策略研究进展 珠蛋白链改变或与珠蛋白合成减少有关的那些。在……里面 这项建议我们的目标是(1)伽玛链的组装与链形成官能化 人胎儿Hb，(2)泛素介导的过量非α珠蛋白的降解 体内的链，以及(3)具有低氧亲和力和 Rb S聚合的阻聚性能。长期目标是 寻找和设计用于镰刀基因治疗的最佳Rb F变异体 细胞病(SCD)和地中海贫血。在具体目标(1)中，我们将测试两个 相关假说；(I)折叠的α-珠蛋白链与中间体组装 在释放之前或之后不久折叠的新生伽马链 多聚核糖体。(Ii)已显示的G-10、14和18位氨基酸 经X-射线单晶分析确定为位于G上的α1-γ1相互作用位 螺旋，对于在体内组装α-和伽马-珠蛋白链以及 在试管中。在特定目的(2)中，我们假设纯化的非α链 四聚体，如Hb杂四聚体，不是泛素化的底物 由于Beta4和Gamma4的结构与Alpha2Beta2异四聚体非常相似 结构。使用兔网织红细胞无细胞系统，我们将测量 在翻译过程中，在没有链的情况下非α链的退化 泛素的存在。在具体目标(3)中，我们将继续调查 Hb F对Hb S聚合的抑制机理 变种(例如，Hb F Gamma 73His、Rb F Gamma 6Val和73His)可以被设计成具有 抑制性能超过Hb F，我们将寻找这样的变体。 我们还将继续寻找比Hb更低氧亲和力的Rb F变体 S不仅通过增强2，3-BPG的相互作用，还通过增强氨基酸 G螺旋上alyL相互作用部位的取代。因为他们的 较低的氧亲和力，这些血红蛋白变体除了有 抗成核性能将在较低水平有效地抑制镰刀状 比原生RB F更低，比如大约10%对20%。理解 伽马和阿尔法链的组装和过剩的降解机制 珠蛋白将为确定最合适的伽马链突变体提供基础 基因疗法，这应该是一种可以通过病毒载体引入的疗法 明显低于原生RbF。此外，这些研究将 对研究蛋白质生物合成和意志的研究人员普遍感兴趣 帮助确定为什么某些突变的珠蛋白链被结合到血红蛋白中 比野生型链更有效或更不有效，以及如何分别 转译的α链和非α链在血红蛋白形成过程中的质量控制 以保存有功能的红细胞。

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.