QUINARY INTERACTIONS OF HEMOGLOBIN S & DESIGN OF SUPER-ANTISICKLING HEMOGLOBINS

（改编自申请人的摘要）脱氧HbS的聚合，尽管 瓦尔-6（Beta）的主要结果涉及合作参与 其他分子间的接触点。潜在的能力，引进和 在适当的靶细胞中表达基因增加了人们对 镰状细胞病的基因治疗一种策略是表达 的抗镰状血红蛋白，以扰乱在 聚合物的具有超抑制活性的抗镰状血红蛋白的设计 潜在的（与pardigmatic HbF相比）可以实现临床 低水平表达的好处。初步研究表明， 表明小鼠，特别是小鼠的非人α链， 猪强烈抑制聚合的互补效应， 序列差异（连锁多位点扰动）。调查人员 假设有可能通过选择 跨物种，最好的连锁多位点序列的差异。的 研究者已经产生了含有非人α-HbS的嵌合HbS， 链以及显示多个序列的嵌合α链 差异以及表现出多个序列的嵌合α-链 差异（与人类相比），旨在映射这种链接的多位点 扰动α-珠蛋白链的模块化构建（使用 最少两个和最多五个模块）通过蛋白酶介导的 人和/或非人α-半乳糖苷酶的互补片段的剪接 链将被用作产生嵌合α-链的主要方法。 猴、马、小鼠和猪的α-链含有4、18、19和 22个序列差异，分别被选择用于初始的 问题研究这些嵌合链将用于定义内- 四聚体功能互补序列差异 引入到顺式和反式二聚体中以增加对 聚合法将测试嵌合血红蛋白的构象 可能改变其功能的差异。超级抑制性α- 链将与具有序列差异的BetaA链杂交， 受体袋区域和/或轴向接触区域 抗镰状化Hb的抑制作用。调查员项目 这种超级抑制性血红蛋白的基因结构可能是一种 镰状细胞病基因治疗新设备 疾病

(Adapted from Applicant's Abstract) Polymerization of deoxy HbS, although a primary consequence of Val-6(Beta), involves a cooperative participation other intermolecular contact sites. The potential ability to introduce and express genes in appropriate target cells has increased the interest in gene therapy of sickle cell disease. One strategy involves the expression of anti-sickling hemoglobins to perturb the lateral contact sites in the polymer. Design of anti-sickling hemoglobins with super inhibitory potential (compared to the pardigmatic HbF) could realize clinical benefits at low levels of expression. The preliminary studies have demonstrated that non-human alpha-chains of mouse and, in particular, of swine strongly inhibit polymerization by complementary effects of many sequence differences (linked multi-site perturbations). The investigators hypothesize that it is possible to optimize this phenomenon by selecting across species, the best linked multi-site sequence differences. The investigators have generated chimeric HbS containing non-human alpha- chains as well as chimeric alpha-chains exhibiting multiple sequence differences The well as chimeric alpha-chains exhibiting multiple sequence differences (compared with human), designed to map such linked multisite perturbations. The modular construction of alpha-globin chains (using a minimum of two and a maximum of five modules) through protease mediated splicing of the complimentary segments of human and/or non-human alpha- chains will be used as the primary approach to generate chimeric a-chains. The a-chains of monkey, horse, mouse, and swine containing 4, 18, 19 and 22 sequence differences, respectively, have been chosen for the initial studies. These chimeric chains will be used to define the intra- tetrameric functional complementarity of the sequence differences introduced into the cis and trans dimers to increase the inhibition of polymerization. Chimeric hemoglobins will be tested for conformational differences that could alter their function. The super-inhibitory alpha- chains will be hybridized with BetaA chains with sequence differences in the acceptor pocket region and/or the axial contact regions to enhance the inhibitory influence of the anti-sickling Hb. The investigators project that gene constructs of such super-inhibitory hemoglobins could be a welcome addition to the armamentarium for the gene therapy of sickle cell disease.