HOMOLOGOUS RECOMBINATION BY SMALL DNA FRAGMENTS

该项目的最终目标是开发一种通过造血干细胞中的靶向基因置换来纠正镰状细胞β-珠蛋白（β/S）序列的方法。镰状细胞性贫血在非洲每64名黑人中就有1人患病，在美国每200至400名非洲裔美国人中就有1人患病。这种疾病是由人β-珠蛋白基因第六密码子中的A>T颠换引起的，该颠换导致蛋白质中Glu被瓦尔取代。许多疗法已经导致归因于突变的病理的部分改善。然而，在基因治疗方面做得很少。小片段同源替换（SFHR），在我的实验室开发的策略，将被用来直接取代内源性β-珠蛋白序列。在囊性纤维化组织和模型报告基因系统中的研究结果表明，可以实现靶向置换。拟议研究的范围将是建立、优化和定义基于SFR的基因替代疗法对纠正血液病的有用性。携带人11号染色体的培养小鼠红白血病（MEL）细胞、表达正常人β-珠蛋白（β/A）的人K562红白血病细胞以及携带人β/S-珠蛋白的小鼠胚胎干细胞和转基因小鼠将为这些研究提供模型系统。表达β/A-珠蛋白的细胞将用突变基因组DNA片段转染，然后在DNA、RNA和蛋白质水平上测定是否存在引入序列。携带β/S-珠蛋白的细胞克隆将被分离和表征，并用于其他SFHR介导的基因靶向。将使用基于脂质的载体转染动物。通过使用等位基因特异性寡核苷酸（阿索）对DNA和mRNA衍生的cDNA进行聚合酶链反应（PCR）扩增，并通过采用限制性片段长度多态性（RFLP）分析的Southern杂交，确定基因置换是否成功。转基因小鼠模型使得有可能评估突变是否可以在体内纠正。将测定小鼠是否存在正常人血红蛋白，因为其与小鼠不同。最终，SFHR基因靶向研究将在CD 34+人造血干细胞中进行。

The ultimate goal of this project is to develop a means to correct sickle cell beta-globin (beta/S) sequences by targeted gene replacement in hematopoietic stem cells. Sickle cell anemia afflicts as many as 1 in 64 blacks in African and from 1 in 200 to 1 in 400 African Americans in the US. The disease is caused by an A>T transversion in the sixth codon of the human beta-globin gene that results in a Glu to Val substitution in the protein. Numerous therapies have led to partial amelioration of the pathology attributed to the mutation. However, very little has been done in the way of gene therapy. Small fragment homologous replacement (SFHR), a strategy developed in my laboratory, will be used to directly replace endogenous beta-globin sequences. Results from studies in cystic fibrosis tissue and in model reporter gene systems indicate that targeted replacement can be achieved. The scope of the proposed studies will be to establish, optimize and define the usefulness of SHFR-based gene replacement therapy to correct hematologic disorders. Cultured mouse erythroleukemia (MEL) cells that carry human chromosome 11, human K562 erythroleukemia cells that express normal human beta-globin (beta/A) as well as mouse embryonic stem cells and transgenic mice that carry human beta/S-globin will provide model systems for these studies. Cells expressing beta/A-globin will be transfected with mutant genomic DNA fragments and then assayed for the presence of incoming sequences at the level of DNA, RNA, and protein. Clones of cells carrying the beta/S-globin will be isolated and characterized, and used for other SFHR-mediated gene targeting. Animals will be transfected using lipid- based vehicles. Successful gene replacement will be determined by polymerase chain reaction (PCR) amplification of DNA and mRNA- derived cDNA with allele-specific oligonucleotides (ASO) and by Southern hybridization employing restriction fragment length polymorphic (RFLP) analysis. The transgenic mouse model makes it possible to assess if the mutation can be corrected in vivo. The mouse will be assayed for the presence of normal human hemoglobin since it distinct from the mouse. Ultimately, SFHR gene targeting studies will be carried out in CD34+ human hematopoietic stem cells.