The ability to alter specifically the genome of cells holds great promise for gene therapy, treatment of certain infectious diseases, and the creation of animal models of human genetic disease. We will develop a general method of targeted mutagenesis capable of introducing a specific, single basepair alteration into any gene in a living cell using the E. coli RecA protein and a synthetic oligonucleotide. The E. coli RecA protein plays an essential role in bacterial homologous recombination and DNA repair and promotes pairing and strand invasion between RecA-coated, single-stranded DNA and homologous duplex DNA in vitro. Here, a modified RecA (engineered to contain a nuclear localization signal) is complexed with a short mutagenic oligonucleotide that contains a single base difference from that found in the target cellular gene. This nucleoprotein complex is introduced into cultured cells using liposomes, where RecA protects the mutagenic oligonucleotide from degradation, directs it toward the nucleus, promotes the searching and pairing of the oligonucleotide with its homologous gene, and initiates strand exchange. Mismatch distortion within the heteroduplex stimulates DNA repair and gene conversion, resulting in stable introduction into the cellular gene of the sequence alteration present on the oligonucleotide. Phase I will examine RecA/oligonucleotide-dependent targeted repair of an extrachromosomal mutant reporter gene in human and primate cultured cells. The efficiency of gene repair will be monitored qualitatively and quantitatively.
This research is directed towards developing a general method of introducing specific genetic alterations into cells. Potential applications include in vivo and ex vivo human gene therapy, therapeutic intervention for certain cancers and infectious diseases, alteration of cells for structural/functional protein analysis, and development of animal models for human genetic disease.
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