HBF Variants for Gene Therapy of Sickle Cell Disease

用于镰状细胞病基因治疗的 HBF 变体

• 批准号: 6734176
• 负责人: KAZUHIKO ADACHI
• 金额: $ 34万
• 依托单位: CHILDREN'S HOSP OF PHILADELPHIA
• 依托单位国家: 美国
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-04-01 至 2006-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6734176
• 关键词: Retroviridae; animal tissue; cell free system; electron microscopy; erythrocytes; fluorescent dye /probe; gene therapy; genetic manipulation; genetic translation; hemoglobin F; hemoglobin Ss; inhibitor /antagonist; low angle X ray diffraction analysis; mutant; polymerization; protein biosynthesis; protein degradation; protein folding; protein protein interaction; protein structure; sickle cell anemia; site directed mutagenesis; thalassemia; thermodynamics; transfection /expression vector; ubiquitin

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.

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 以及如何从其中去除错误折叠和不稳定的未组装珠蛋白链 红细胞未知。此外，Hb F 的基本机制 抑制聚合并改善 SCD 的临床病程并非如此 完全明白了。阐明此类机制有助于 开发基因治疗疾病的策略 改变的球蛋白链或与球蛋白合成减少相关的球蛋白链。在 该提案我们的目标是（1）用链组装伽玛链以形成功能性 人胎儿 Hb，(2) 泛素介导的过量非 α 珠蛋白降解 (3) 具有低氧亲和力的工程 Hb F 变体 对 Rb S 聚合的抑制性能。长期目标是 识别和设计用于镰状细胞病基因治疗的最佳 Rb F 变体 细胞疾病（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-珠蛋白链与中间体组装 在释放之前或之后不久折叠新生的伽玛链 多聚核糖体。 (ii) G-10、14和18位的氨基酸，已显示 通过 X 射线晶体学分析位于 G 上的 alpha1gamma1 相互作用位点 螺旋，对于体内 α 和 γ 珠蛋白链的组装以及 体外。在特定目标 (2) 中，我们假设纯化的非 α 链 四聚体，如 Hb 异源四聚体，不是泛素化的底物 因为 Beta4 和 gamma4 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 在翻译过程中没有链的情况下非α链的降解 泛素的存在。在具体目标（3）中，我们将继续研究 Hb F 抑制 Hb S 聚合的机制。我们假设 Hb F 可以设计变体（例如，Hb F gamma 73 His、Rb F gamma 6Val 和 73 His） 抑制特性超过 Hb F，我们将寻求此类变体。 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，还通过增强氨基酸 G 螺旋上烯丙基相互作用位点的取代。因为他们的 较低的氧亲和力，这些血红蛋白变体除了具有 抗成核特性可有效抑制较低水平的镰状形成 比天然 Rb F 低，例如约 10% 对 20%。的理解 γ链和α链的组装以及过量的降解机制 球蛋白将为确定最合适的伽马链突变体提供基础 用于基因治疗，应该是可以通过病毒载体引入的基因治疗 的水平明显低于天然 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并将 帮助确定为什么一些突变珠蛋白链会并入血红蛋白中 比野生型链或多或少有效，以及如何分开 翻译的α链和非α链在血红蛋白形成过程中受到质量控制 以保存功能性红细胞。