HOMOLOGOUS RECOMBINATION BY SMALL DNA FRAGMENTS

University Of California San Francisco, San Francisco CA

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Abstract

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.