CORRECTION OF THE SICKLE CELL MUTATION BY GENE TARGETING

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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.