HBF VARIANTS FOR GENE THERAPY OF SICKLE CELL DISEASE

描述：这项拟议的研究涉及以下抑制机制： 通过HbF聚合HbS，红色中HbF的高氧亲和力的机理 血细胞的α和γ链的组装机制， 血红蛋白形成胎儿血红蛋白使用各种生化和 生物物理方法。 利用这些研究的结果， 研究人员希望设计新的抗镰状化HbF变体， 用于镰状细胞病的基因治疗。 在具体目标1中，氨基酸 将鉴定HbF中对FS杂交至关重要的残基 阵 共聚物的初步X射线分析表明，Val-β HbS中的6与相邻分子中的受体口袋连通， Thr-β 87通过一个界面连接的桥梁发挥重要作用， 增强疏水相互作用的水。 进一步研究 涉及beta和gamma 87。 具体目标2中，研究将 以表征Hb F的氧结合和聚合特性 和S混合物。 这些研究的目的是确认氧分压差 F突变体与2，3-DPG的结合效果与HbA相似。 在 初步研究，已经产生了这样的突变体（HbF γ G1 V，E5 P， S143H）。 这一目标的研究将进一步表征这些突变体， 包括X射线分析、氧结合研究和共聚 测量. 在具体目标3中，将开展研究， α和γ链组装形成HbF以及 突变体γ链对α和β-S链组装的影响。 突变体 提出这将导致理解的残留物， 对于理解这些组件的速率控制至关重要， 总体目标是提高HSF混合血红蛋白的效率。 这些突变体的特定位置将在链界面处， 还有γ链表面电荷。 在第四个目标中，研究人员 建议利用前三个目标的成果，设计新颖的 HbF变体，其将具有最佳的氧亲和力和 与β-S链共组装以形成HbFS杂合体。 的最终目标 其目的是为患者的基因治疗指定最佳基因产物， 镰状细胞性贫血 在这方面，调查人员还指出， 有效表达是必不可少的。

DESCRIPTION: This proposed research addresses the inhibitory mechanisms of HbS polymerization by HbF, mechanisms of high oxygen affinity of HbF in red blood cells, and the mechanism of assembly of alpha and gamma chains of hemoglobin to form fetal hemoglobin using a variety of biochemical and biophysical methods. Using the results from these studies, the investigators hope to design novel HbF anti-sickling variants for eventual use in gene therapy in sickle cell disease. In Specific Aim 1, amino acid residues in HbF will be identified which are critical for FS hybrid formation. Preliminary X-ray analyses of copolymers indicates that Val-beta 6 in HbS communicates with the acceptor pocket in the adjacent molecule and that Thr-beta 87 plays an important role through a bridge of interfacial water which strengthens the hydrophobic interaction. Further studies involving beta and gamma 87 are proposed. In Specific Aim 2, studies will be done to characterize oxygen binding and polymerization properties of Hb F and S mixtures. The goal of these studies is to confirm differential oxygen binding of F mutants with 2,3-DPG effects similar to those of HbA. In preliminary studies, such mutants have been produced (Hb F gamma G1V, E5P, S143H). Studies in this aim will further characterize these mutants, including X-ray analyses, oxygen binding studies and copolymerization measurements. In Specific Aim 3, studies will be undertaken to characterize the assembly of alpha and gamma chains to form HbF as well as the effect of mutant gamma chains on the assembly of alpha and beta-S chains. Mutants are proposed which will lead to the understanding of the residues which are critical to the understanding of the control of rates of these assemblies, with the overall goal to increase the efficiency of HSF hybrid hemoglobin. Specific locations for these mutants will be at the chain interfaces, but also gamma chain surface charge. In Specific Aim 4, the investigators propose to use the results of the first three aims to design new and novel HbF variants which will have optimal properties of oxygen affinity and coassembly with beta-S chains to form HbFS hybrids. The ultimate goal of this aim is to specify the optimal gene product for gene therapy in patients with sickle cell anemia. In that regard, the investigators also point out that efficient expression is essential.