OLIGONUCLEOTIDE BASED GENE THERAPY FOR SICKLE CELL ANEMIA

（改编自申请人的摘要）镰状细胞性贫血是 一种由单碱基对突变引起的遗传疾病的原型， 人类珠蛋白基因第六密码子中的A到T颠换。以低 氧张力，取代一个单一的氨基酸（GLU>瓦尔），在 血红蛋白的β-珠蛋白亚基导致HB S的聚合， 导致红细胞形状不规则。镰状红细胞 被困在微循环中，造成极度疼痛和损害， 多个器官 研究人员提出要检验三链体形成的假设， 与诱变剂连接的寡核苷酸可用于产生 β S和γ-珠蛋白基因突变，以抑制 HB S在红细胞内，这可能用于基因治疗， 镰状细胞病携带靶基因的基于SV 40的穿梭载体 将建立一个改进的哺乳动物细胞突变测定系统， 开发以促进三重定向诱变的研究， 体内的基因;与靶位点结合的寡核苷酸将被 设计和合成;寡核苷酸特征（如 核苷酸组成、化学修饰和类似物取代） 并检测靶向诱变。最后，实验将 进行了直接测试的假设，有针对性的诱变， 三链体形成寡核苷酸介导的β S和γ-珠蛋白基因 可以在哺乳动物细胞的染色体DNA中体内实现， 文化 最终目标是将诱变寡核苷酸递送至骨 骨髓细胞和永久性和遗传性突变的引入 插入β S和/或γ-珠蛋白基因的所需位点， 细胞内Hb S的聚合将被抑制。尽管它可能是 在这项技术应用于临床之前， 应用程序提出了一个机构的工作，以建立潜在的 靶向诱变在临床和科学研究中的作用。

(Adapted from the Applicant's Abstract) Sickle cell anemia is the prototype of a genetic disease caused by a single base-pair mutation, an A-to-T transversion in the sixth codon of the human globin gene. At low oxygen tensions, the substitution of a single amino acid (GLU>Val) in the beta-globin subunit of hemoglobin results in a polymerization of HB S and leads to irregular shaped erythrocyte cells. The sickled erythrocyte cells become trapped n the microcirculation, causing extreme pain and damage to multiple organs. The investigators propose to test the hypothesis that triplex-forming oligonucleotides linked to mutagenic agents can be used to generate mutations in betaS and gamma-globin genes to inhibit the polymerization of HB S within erythrocyte cells and this may be utilized in gene therapy for sickle cell diseases. SV40-based shuttle vectors carrying the target genes will be constructed; an improved mammalian cell mutation assay system will be developed to facilitate the study of triplex-directed mutagenesis of the genes in vivo; oligonucleotides that bind to target sites will be designed and synthesized; oligonucleotide characteristics (such as nucleotide composition, chemical modifications, and analog substitutions) and targeted mutagenesis will be examined. Finally, experiments will be carried out to direct test the hypothesis that targeted mutagenesis of betaS and gamma-globin genes mediated by triplex-forming oligonucleotides can be achieved in vivo in the chromosomal DNA of mammalian cells in culture. The ultimate goal is the delivery of mutagenic oligonucleotides to bone marrow cells and the introduction of permanent and inheritable mutations into desired sites of the betaS and/or gamma-globin gene so that the polymerization of Hb S within cells will be inhibited. Although it may be some time before this technology is applied clinically, this grant application proposes a body of work to establish the potential for the role of targeted mutagenesis in both clinical and scientific endeavors.