CORRECTION OF THE SICKLE CELL MUTATION BY GENE TARGETING

描述(直接取自申请人的摘要) 镰状细胞性贫血是一种遗传代谢性疾病， 非洲黑人的比例为1.6%，非洲裔美国人的比例从0.5%上升到0.25% 美国。这种疾病是由A&GT；T基因错位引起的 人类β-珠蛋白基因的第六个密码子，导致Glu to Val 蛋白质中的替代。表型上，有一种聚合体 导致无数种病理的血红蛋白最终 会导致个体的死亡。无数的治疗方法导致了 由于突变而引起的致病效应的改善， 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)，一种基因 依赖小片段同源替换的治疗策略 基因组DNA以纠正给定基因中的突变。我们的结果表明 对突变基因的纠正可以在DNA、RNA和 人上皮细胞的功能水平。因为这种方法具有 通过保留基因来保持基因完整性的明显优势 转录基因与内源启动子之间的关系， 这比利用cDNAs表达载体的cDNA法更可取 异源启动子的调控。这里提出的研究有 两个目标：1)在培养的细胞中引入镰状细胞突变 携带人类11号染色体的小鼠红白血病(MEL)细胞和 表达人β-珠蛋白；2)小鼠的分离和转染 造血祖细胞。将小分子病毒导入细胞 基因组DNA片段，然后检测是否存在传入的 DNA、RNA和蛋白质水平的序列。MEL细胞的克隆 携带β/S珠蛋白的人将被分离并研究纠正 还将进行突变。成功的基因替换将是 通过聚合酶链式反应(PCR)扩增DNA和 用等位基因特异性寡核苷酸(ASO)和 利用限制性片段长度多态的Southern杂交 (RFLP)分析。此外，如果造血细胞表达 分离出血红蛋白后，可以分析其蛋白质的存在 正常的血红蛋白。在这些研究的下一阶段，我们将尝试 纠正携带人β/S珠蛋白转基因小鼠的突变 轨迹。因为正常的人类血红蛋白与小鼠不同，它可以 很容易被化验。最终，这一策略可以应用于纠正 人外周血造血细胞中的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.