Most current gene therapy approaches are based on a """"""""gene addition"""""""" strategy, where a functional transgene cassette is delivered to cells and expressed from episomal or randomly integrated molecules. A potentially more powerful approach would be to correct mutations at their normal chromosomal locations. The major advantages of this """"""""gene correction"""""""" approach include properly regulated gene expression and the removal of dominant disease-causing mutations. Until recently, therapeutic gene correction was felt to be beyond the scope of genetic technologies. The applicant's recent demonstration that adeno-associated virus (AAV) vectors can be used to introduce specific genetic modifications into homologous human chromosomal loci at high frequencies (nearly 1 percent of normal human fibroblasts) suggests that this strategy may ultimately be efficient enough to allow for therapeutic gene correction. The experiments described in this proposal will explore several aspects of gene correction by AAV vectors, in an attempt to optimize gene targeting rates and improve our understanding of the mechanisms involved. AAV vectors will be used to introduce specific modifications into both normal human genes (such as HPRT) and engineered chromosomal target sites containing mutant marker genes introduced by retroviral vectors. Targeted chromosomal loci will be analyzed by sequencing recovered plasmids containing corrected genes to completely define the structure of the targeted loci. Different types of mutations (insertions, deletions and mismatches) will be corrected and the length of homology between the vector and chromosomal target locus will be adjusted to optimize vector design. Cellular factors important for the gene targeting reaction will be analyzed by varying cell division rates, treating cells with agents that induce DNA repair functions, and measuring gene correction rates in cells with mutations in DNA repair genes. Transgenic and mutant mouse models will be used to study AAV- mediated gene targeting in vivo, including gene correction at beta-glucuronidase and beta-galactosidase genes, which will be assayed by histochemical staining of tissue sections and sequencing of corrected loci recovered as plasmids in bacteria. These experiments should help define the basic biology of gene targeting by AAV vectors, and determine its potential for scientific and therapeutic applications.
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