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: 2901340
• 负责人: KAZUHIKO ADACHI
• 金额: $ 43.51万
• 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 2002-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2901340
• 关键词: DNA binding protein; Retroviridae; aminoacid biosynthesis; biophysics; circular dichroism; erythrocytes; gene expression; gene induction /repression; gene mutation; gene therapy; genetic manipulation; hematopoietic stem cells; hemoglobin F; hemoglobin Ss; human tissue; inhibitor /antagonist; intermolecular interaction; low angle X ray diffraction analysis; oxygen; polymerization; recombinant virus; sickle cell anemia; site directed mutagenesis; transfection /expression vector

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.

描述：这项拟议的研究涉及 HBF聚合HBF，HBF高氧亲和力的机制 血细胞，以及α和伽马链组装的机理 血红蛋白使用多种生化和 生物物理方法。 利用这些研究的结果， 调查人员希望设计新颖的HBF反踢式变体用于最终 用于镰状细胞疾病中的基因疗法。 在特定的目标1中，氨基酸 将确定HBF中的残留物，这对于FS杂种至关重要 形成。 共聚物的初步X射线分析表明Val-Beta 6在HBS中与相邻分子中的受体口袋进行通信 Thr-Beta 87通过界面桥起着重要的作用 加强疏水相互作用的水。 进一步的研究 提出了涉及Beta和Gamma 87。 在特定的目标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差分氧 F具有2,3-DPG效应的F突变体的结合类似于HBA。 在 初步研究，已经产生了此类突变体（Hb F gamma g1v，e5p， S143H）。 以此目的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。 在特定目标3中，将进行研究以表征 Alpha和Gamma链的组装形成HBF，以及 突变的伽马链在α和β-S链组装上。 突变体是 提出的将导致对残留物的理解 对于理解这些组件速率的控制至关重要， 总体目标是提高HSF杂交血红蛋白的效率。 这些突变体的特定位置将位于链界面，但 还有伽马链表面电荷。 在特定目标4中，调查人员 建议使用前三个目标的结果来设计新的和新颖的 HBF变体将具有氧亲和力的最佳特性和 与β-S链结合以形成HBFS杂种。 最终目标 该目的是指定患者基因治疗的最佳基因产物 镰状细胞贫血。 在这方面，调查人员还指出 该有效的表达是必不可少的。