HOMOLOGOUS RECOMBINATION BY SMALL DNA FRAGMENTS

该项目的最终目标是通过在造血干细胞中靶向基因替换来开发一种纠正镰状细胞β -球蛋白（β /S）序列的方法。在非洲，镰状细胞性贫血的发病率高达64分之一，而在美国，这一比例从200分之一到400分之一不等。这种疾病是由人类-珠蛋白基因的第6个密码子的A>T翻转引起的，导致蛋白质中的Glu到Val替换。许多治疗已经导致部分改善病理归因于突变。然而，在基因治疗方面做得很少。小片段同源替代（SFHR）是我实验室开发的一种策略，将用于直接替代内源性β -珠蛋白序列。囊性纤维化组织和模型报告基因系统的研究结果表明，靶向替代是可以实现的。拟议研究的范围将是建立、优化和确定基于shfr的基因替代疗法对纠正血液病的有效性。培养携带人类11号染色体的小鼠红白血病（MEL）细胞、表达正常人类β -珠蛋白（β /A）的人K562红白血病细胞以及小鼠胚胎干细胞和携带人类β / s -珠蛋白的转基因小鼠将为这些研究提供模型系统。表达β / a -珠蛋白的细胞将被转染突变的基因组DNA片段，然后在DNA、RNA和蛋白质水平上检测传入序列的存在。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。动物将使用脂质载体进行转染。成功的基因替换将通过DNA和mRNA衍生的cDNA的聚合酶链反应（PCR）扩增和限制性片段长度多态性（RFLP）分析来确定。转基因小鼠模型使评估是否可以在体内纠正突变成为可能。小鼠将被检测是否存在正常的人血红蛋白，因为它与小鼠不同。最终，SFHR基因靶向研究将在CD34+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.