HOMOLOGOUS RECOMBINATION BY SMALL DNA FRAGMENTS

该项目的最终目标是开发一种通过定向基因替换在造血干细胞中纠正镰状细胞β-珠蛋白(β/S)序列的方法。在非洲，黑人中有1人患有镰状细胞性贫血，在美国，每200到400名非洲裔美国人中就有1人患有镰状细胞性贫血。这种疾病是由人类β-珠蛋白基因第六密码子的A&gT；T颠倒引起的，导致蛋白质中的Glu到Val的替换。许多治疗方法已经导致了这种突变所致的病理部分改善。然而，在基因治疗方面所做的工作很少。小片段同源替换(SFHR)是我实验室开发的一种策略，将被用于直接替换内源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，可以实现有针对性的替代。拟议研究的范围将是建立、优化和确定基于SHFR的基因替代疗法在纠正血液病方面的有用性。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、小鼠胚胎干细胞和携带人β/S-珠蛋白的转基因小鼠将为这些研究提供模型系统。将突变的基因组DNA片段导入表达β/A-珠蛋白的细胞，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鉴定，并用于其他SFHR介导的基因靶向。动物将使用基于脂质的载体进行转基因。成功的基因替换将通过等位基因特异性寡核苷酸(ASO)的聚合酶链式反应(PCR)扩增DNA和信使核糖核酸(MRNA)以及采用限制性片段长度多态性(RFLP)的Southern杂交来确定。转基因小鼠模型使评估突变能否在体内得到纠正成为可能。小鼠将被检测是否存在正常的人类血红蛋白，因为它与小鼠不同。最终，将在CD34+的人造血干细胞中进行SFHR基因靶向研究。

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.