OLIGONUCLEOTIDE BASED GENE THERAPY FOR SICKLE CELL ANEMIA

(改编自申请人摘要)镰状细胞性贫血是 由单个碱基对突变引起的遗传病的原型 人类珠蛋白基因第六密码子的A-T颠换。处于低谷 氧分压，单一氨基酸(GLU&GT；Val)的取代 血红蛋白β-珠蛋白亚基导致血红蛋白S和 导致不规则形状的红细胞。镰刀状的红细胞 被困在微循环中，造成极度疼痛和损害 多个器官。 研究人员提议检验三链形成的假说 与诱变剂相连的寡核苷酸可以用来产生 β基因和γ-珠蛋白基因突变抑制血管内皮细胞的聚合 Hb S在红细胞内的表达，这可能会被用于基因治疗 镰状细胞病。基于SV40的携带目的基因的穿梭载体 将被构建；一个改进的哺乳动物细胞突变检测系统将 是为了便于研究三重定向诱变 体内的基因；与靶点结合的寡核苷酸将是 设计和合成的；寡核苷酸特征(如 核苷酸组成、化学修饰和类似物替代) 并将对定向突变进行检查。最后，实验将是 进行直接测试的目的是为了直接测试以下假设 三链形成寡核苷酸介导的β-和γ-珠蛋白基因 可以在体内实现在哺乳动物细胞的染色体DNA中 文化。 最终的目标是将诱变的寡核苷酸输送到骨骼 骨髓细胞与永久性和可遗传突变的引入 进入Betas和/或伽马-珠蛋白基因的所需位置，以便 Hb S在细胞内的聚合被抑制。尽管它可能是 在这项技术应用于临床之前的一段时间，这项拨款 应用程序提出了一系列工作，以建立 靶向突变在临床和科学工作中的作用。

(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.