A novel strategy for gene therapy has been developed and shown promise as a potential therapeutic for inherited disorders. Unique synthetic molecules, known as chimeric oligonucleotides, are now in the pipeline toward clinical trials. Recent reports from the investigator's and other laboratories have described the use of chimeric RNA/DNA oligonucleotides to introduce targeted base pair substitutions into episomal or genomic DNA of mammalian cells in culture or in animals. With a frequency of targeted mutagenesis greater than 1 percent, chimeraplasty represents a promising new approach to potentially correct genetic defects attributable to point or frameshift mutations. In this grant, the investigators propose to study the mechanism of gene correction relying upon studies with purified enzymes and with human cell-free extracts. Preliminary work indicates that correction proceeds by a two step pathway consisting of homologous recombination of the chimeric oligonucleotide with dsDNA followed by resolution and repair of the mismatched joint molecule. In the repair step the target DNA acquires the sequence of the chimera thus leading to correction. The investigators will use standard in vitro assays to investigate how RecA and hsRad51/52 proteins catalyze homologous pairing and strand exchange between chimeras and homologous double-stranded DNA targets. This reaction is believed to generate a novel joint molecule with a complement-stabilized displacement loop. The properties of this joint molecule would be studied at both the biochemical and biophysical levels. Subsequent processing of the joint molecule to yield a corrected and re-paired DNA target will be investigated using both wild-type and repair deficient human cell-free extracts. In preliminary work, we have demonstrated that a wild-type extract catalyzes chimera-mediated correction of mutant antibiotic resistance genes help elucidate the correction pathway will be carried out using the extract as a source of enzymatic activity. Assays will involve both genetic and biochemical readouts. In parallel, large numbers of chimeric oligonucleotides that systemically differ in structure will be screened for activity. Preliminary results indicate that the all DNA strand directs the correction, while the strand containing RNA stabilizes the chimera-target complex. These modified chimeras, which increase the efficiency of the reaction in a cell-free extract, also elevate the frequency in cultured cells. Hence, biochemical results translate into whole cells.
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