HBF VARIANTS FOR GENE THERAPY OF SICKLE CELL DISEASE

描述：这项拟议的研究解决了 HBF引发的HBS聚合及HBF在红色中的高亲和力机理 血细胞，以及α和伽马链的组装机制 血红蛋白利用多种生物化学方法形成胎儿血红蛋白 生物物理学方法。利用这些研究的结果， 调查人员希望设计出新的HBF抗病变种 用于镰状细胞病的基因治疗。在特定目标1中，氨基酸 将确定HBF中对FS杂交种至关重要的残留物 队形。共聚物的初步X射线分析表明，Val-beta HBS中的6与相邻分子中的受体口袋通信，并 Thr-β87通过界面桥梁发挥重要作用 加强疏水相互作用的水。进一步研究 涉及贝塔和伽马87。在具体目标2中，研究将 对Hb F的氧键和聚合性能进行了表征 和S的混合物。这些研究的目标是确认不同的氧气 F突变体与2，3-DPG的结合效果与HBA相似。在……里面 初步研究，已经产生了这样的突变体(Hb F Gamma G1V，E5P， S143H)。这一目标的研究将进一步描述这些突变体的特征， 包括X射线分析、氧结合研究和共聚 测量。在具体目标3中，将进行研究，以确定 α链和伽马链的组装形成HBF以及 突变的伽马链上组装的α和β-S链。变种人是 建议，这将导致对残留物的理解 对理解这些组件的速率控制至关重要， 总体目标是提高HSF杂化血红蛋白的效率。 这些突变体的特定位置将在链界面上，但 也叫伽马链表面电荷。在具体目标4中，调查人员 建议利用前三个目标的结果来设计新的和新颖的 HBF变体将具有最佳的氧气亲和力和 与β-S链共组装形成HbFS杂交物。的最终目标是 这一目的是为了确定用于患者基因治疗的最佳基因产物。 患有镰状细胞性贫血。在这方面，调查人员还指出， 这种高效的表达是必不可少的。

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.