Gene Targeting and Random Integration by Adeno-Associated Virus (AAV) Vectors. AAV vectors can be used to efficiently modify homologous chromosomal sequences in normal mammalian cells by a gene targeting process. This approach introduces precise genetic changes at specific chromosomal loci. Gene targeting is superior to """"""""gene addition"""""""" strategies normally used in gene therapy, as it preserves the normal transcriptional control of the target locus, and in principle can avoid insertional mutagenesis due to random integration. Although current gene targeting frequencies of AAV vectors are high (1% of normal human cells), improvements will be necessary to obtain therapeutic targeting levels in many applications. In addition, random integration events can still occur, which complicate the recovery of targeted cells and can lead to unwanted mutations. The long-term objective of this application is to increase the versatility and efficiency of AAV-mediated gene targeting, while avoiding unwanted random integration events. To achieve this goal, the mechanism of gene targeting will be explored by studying its relationship to DNA replication, RNA transcription, and specific cellular factors involved in DNA repair. Improved targeting vectors will be designed to introduce a variety of genetic modifications, and selection procedures will be used to enrich for targeted cells and screen out random integrants. Optimized AAV vectors will be used to carry out gene targeting in animals, including in vivo correction of mutations in mice as a preclinical model of potential human therapies, and the creation of genetically engineered, cloned sheep and cows to study cystic fibrosis and prion diseases respectively. As the potential genotoxicity of AAV vectors will remain a factor in clinical applications involving gene targeting or gene addition, large-scale analyses of random integration will be conducted in normal human cells and mice, to establish where AAV vectors integrate and whether integration carries the risk of activating nearby proto-oncogenes. In summary, this proposal will develop AAV gene targeting vectors for several applications, determine their therapeutic potential, and assess their genotoxic risks. It is hoped that these studies will lead to therapeutic gene targeting protocols for the treatment of genetic and acquired diseases.
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