RESTORATION OF GLOBIN GENE EXPRESSION IN THALASSEMIA

地中海贫血中球蛋白基因表达的恢复

• 批准号: 6125893
• 负责人: RYSZARD KOLE
• 金额: $ 21.35万
• 依托单位: UNIV OF NORTH CAROLINA CHAPEL HILL
• 依托单位国家: 美国
• 财政年份: 1994
• 资助国家: 美国
• 起止时间: 1994-12-01 至 2002-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6125893
• 关键词: RNA splicing; antisense nucleic acid; blood disorder chemotherapy; cell line; disease /disorder model; drug screening /evaluation; erythroid stem cell; gene expression; gene mutation; globin; human genetic material tag; laboratory mouse; liposomes; membrane transport proteins; messenger RNA; nonhuman therapy evaluation; oligonucleotides; polymerase chain reaction; thalassemia; transfection /expression vector; western blottings

The long term goal of this project is application of antisense oligonucleotides and antisense vectors in treatment of thalassemia. The project is based on a unique application of antisense oligonucleotides in which these compounds are used to restore correct functioning of a defective gene rather than to inhibit the expression of an undesirable gene, as commonly applied. Specifically, antisense oligonucleotides are used to reverse aberrant splicing of human beta-globin pre-mRNA by blocking splice sites activated by the mutations in intron 2 of Beta- globin gene which cause thalassemia. The oligonucleotides not only inhibit aberrant splicing but, by forcing the spliceosome to form on the adjacent correct splice sites, restore correct splicing of beta-globin pre-mRNA and in consequence restore translation of beta-globin polypeptide. In the previous granting period the effective correction of splicing in a number of thalassemic mutants was accomplished both in the cell free splicing extracts and in model cell lines which expressed thalassemic beta-globin transcripts. The main thrust of the work proposed in this application will be to establish the conditions for antisense treatment of thalassemic erythroid cells in vitro and in vivo in animal model. The specific aims to accomplish these goals are: 1) To optimize the correction of splicing of human thalassemic beta-globin pre-mRNAs by antisense oligonucleotides in K562 cells expressing IVS2-654, IVS2-705 and IVS2-745 mutants. 2) To optimize the effects of antisense oligonucleotides on human beta-globin expression in bone marrow of the IVS2-654 mouse. 3) To treat the IVS2-654 thalassemic mice with antisense oligonucleotides either in a free form or with the aid of delivery agents. 4) To test in K-562 cell lines the correction of splicing of thalassemic beta-globin pre-mRNA by antisense vectors targeted to the aberrant splice sites. 5) To test the antisense snRNA vectors developed in Specific Aim 4 in IVS2-654 mice. To provide high level of expression and to explore the possibility of tissue tropic delivery of antisense sequences the snRNA genes will be incorporated into adeno-associated virus.

该项目的长期目标是使用反义 寡核苷酸和反义载体治疗thalassymia。 这 项目基于反义寡核苷酸的独特应用 其中使用这些化合物来恢复A的正确功能 有缺陷的基因而不是抑制不良的表达 基因，通常应用。 具体而言，反义寡核苷酸是 用于扭转人β-珠蛋白pre-mRNA的异常剪接 阻止β-内含子2中的突变激活的剪接位点 引起丘脑贫血的球蛋白基因。 寡核苷酸不仅 抑制异常剪接，但通过强迫剪接体在上形成 相邻正确的剪接站点，恢复β-珠蛋白的正确剪接 前MRNA和因此恢复β-珠蛋白的翻译 多肽。 在上一个授予期内有效更正 在许多丘脑血症突变体中的剪接都完成了 无细胞的剪接提取物和表达的模型细胞系 Thalassemic Beta-Globin转录本。 作品的主要力量 在本申请中提出的建议是建立 反义在体外和体内对丘脑血症细胞的治疗 在动物模型中。 实现这些目标的具体目的是：1） 优化校正人类丘脑β-珠蛋白的剪接 在表达K562细胞中的反义寡核苷酸前MRNA IVS2-654，IVS2-705和IVS2-745突变体。 2）优化 骨中人β-珠蛋白表达的反义寡核苷酸 IVS2-654小鼠的骨髓。 3）治疗IVS2-654小鼠 用反义寡核苷酸自由形式或辅助 交付代理。 4）在K-562细胞系中测试校正 反义载体对丘脑β-珠蛋白pre-mRNA的剪接 针对异常的剪接站点。 5）测试反义snRNA 在IVS2-654小鼠中，特定AIM 4开发的矢量。 提供高 表达水平并探索组织热带组织的可能性 反义序列的递送SNRNA基因将合并 进入腺相关病毒。