CORRECTION OF THE SICKLE CELL MUTATION BY GENE TARGETING

描述（直接摘自申请人摘要） 镰状细胞性贫血是一种遗传代谢疾病， 1.6非洲黑人的比例从0.5%到0.25%， 美国的 这种疾病是由A > T颠换引起的， 人类β-珠蛋白基因的第六个密码子，导致Glu变为瓦尔 蛋白质中的取代。 从表型上看， 血红蛋白导致了无数的病理， 导致个人的死亡。 许多疗法导致了 改善归因于突变的致病作用， 然而，在基因治疗领域所做的工作很少。 我们 已经开发出小片段同源重组（SFHR）， 依赖于小片段同源置换的治疗策略 基因组DNA来纠正特定基因的突变。 我们的研究结果表明 突变基因的校正可以在DNA、RNA和 在人类上皮细胞中的功能水平。 因为这种方法具有 通过保留基因的完整性来保持基因的完整性， 转录基因与内源启动子之间的关系， 优选cDNA方法，其利用cDNA表达载体， 异源启动子的调控。 这里提出的研究有 两个目的：1）在培养的镰状细胞中引入镰状细胞突变， 携带人11号染色体的鼠红白血病（MEL）细胞， 表达人β-珠蛋白，和2）分离和转染小鼠 造血祖细胞 细胞将被转染小 基因组DNA片段，然后测定是否存在传入的 DNA、RNA和蛋白质水平上的序列。 MEL细胞克隆 携带β/s球蛋白的人将被分离出来，并研究如何纠正 还将进行突变。 成功的基因替换将是 通过DNA的聚合酶链反应（PCR）扩增测定， 用等位基因特异性寡核苷酸（阿索）和通过 限制性片段长度多态性Southern杂交 （RFLP）分析。 此外，如果造血细胞表达 当分离血红蛋白时，可以分析蛋白质中存在的 血红蛋白正常 在这些研究的下一阶段，我们将尝试 在携带人类β/S-珠蛋白的转基因小鼠中纠正突变 基因座 因为正常人的血红蛋白与老鼠的不同， 易于分析。 最终，该策略可以用于纠正 人造血细胞中的β/S-珠蛋白突变。

DESCRIPTION (Taken directly from applicant's abstract) Sickle cell anemia is a genetic metabolic disease that afflicts as many as 1.6 of blacks in Africa and from .5% to .25% of African Americans in the United States. The disease is caused by an A > T transversion in the sixth codon of the human beta-globin gene, resulting in a Glu to Val substitution in the protein. Phenotypically, there is a polymerization of the hemoglobin that results in a myriad of pathologies that ultimately lead to the death of the individual. Numerous therapies have led to the amelioration of the pathogenic effects attributed to the mutation, however, very little work has been done in the area of gene therapy. We have developed small fragment homologous recombination (SFHR), a gene therapy strategy that relies on homologous replacement by small fragments of genomic DNA to correct mutations in a given gene. Our results indicate that correction of a mutant gene can be achieved at the DNA, RNA, and functional level in human epithelial cells. Because this approach has the obvious advantage of maintaining the integrity of the gene by retaining the relationship between the transcribed gene and the endogenous promoter, it is preferable to cDNA methods that utilize cDNA expression vector under the regulation of heterologous promoters. The studies proposed here have two aims: 1) the introduction of a sickle cell mutation in cultured murine erythroleukemia (MEL) cells that carry human chromosome 11 and express human beta-globin, and 2) the isolation and transfection of mouse hematopoietic progenitor cells. Cells will be transfected with small genomic DNA fragments and then assayed for the presence of incoming sequences at the level of DNA, RNA, and protein. Clones of MEL cells carrying the beta/s globin will be isolated and studies to correct the mutation will also be carried out. 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. In addition, if hematopoietic cells expressing hemoglobin are isolated, the protein can be analyzed for the presence of normal hemoglobin. In the next phase of these studies, we will attempt to correct the mutation in transgenic mice carrying the human beta/s-globin locus. Because normal human hemoglobin is distinct from the mouse, it can be readily assayed. Ultimately this strategy can be applied to correct the beta/s-globin mutation in human hematopoietic cells ex vivo.