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，血红素和球蛋白亚基协调组装的机制 以及从中删除的错误折叠和不稳定的不稳定的环球链链 红细胞尚不清楚。另外，HB F的基本机制 抑制聚合并改善SCD的临床过程不是 完全理解。阐明这种机制可以有助于 制定基因治疗策略在治疗疾病 球蛋白链的改变或与球蛋白合成减少有关的链。在 我们的提议我们针对（1）用链条形成功能的伽马链组件 人类胎儿HB，（2）泛素介导的过量非α球蛋白的降解 体内链和（3）具有低氧亲和力的工程HB F变体 RB S聚合的抑制特性。远程目标是 识别和设计用于镰刀基因治疗的最佳RB F变体 细胞疾病（SCD）和丘脑症。在特定目标（1）中，我们将测试两个 相关假设； （i）折叠的α-珠蛋白链，并与 释放前或不久后折叠起来的新生伽马链 多核糖体。 （ii）G-10、14和18的氨基酸已显示 通过X射线晶体学分析，位于G上的Alpha1gamma1相互作用位置 螺旋对于在体内以及体内的α-和γ-球蛋白链的组装至关重要 体外。在特定目标（2）中，我们假设纯化的非α链 四聚体，例如HB异质四聚体，不是泛素化的底物 由于beta4和gamma4结构与alpha2beta2杂波非常相似 结构。使用兔子网状细胞不含细胞系统，我们将测量 在翻译过程中没有链的情况下，非α链的降解 存在泛素。在特定目标（3）中，我们将继续调查 HB F的Hb S聚合的抑制作用机理。我们假设Hb f 可以设计变体（例如HB F伽马73 HIS，RB F Gamma 6val＆73 HIS）。 超过HB F的抑制特性，我们将寻求这种变体。 我们还将继续寻求氧气亲和力低的RB F变体 不仅通过增强2,3-bpg相互作用，还可以增强氨基酸 在G螺旋上的烷基相互作用位点取代。因为他们 较低的氧亲和力，除了具有 抗核特性将有效地抑制较低水平的疾病 比本地RB F（例如约10％vs. 20％）会。理解 伽马和α链的组装以及过度降解的机制 Globin将为确定最合适的伽马链突变体提供基础 对于基因疗法，应该是病毒载体引入的一种 在显着低于天然Rb F的水平。此外，这些研究将 对于研究蛋白质生物合成并将将 帮助确定为什么将某些突变球蛋白链纳入血红蛋白 比野生型连锁链以及如何分别有效 在血红蛋白形成期间，翻译的α和非α链是质量控制的 保留功能性红细胞。