Gene Targeting and Integration by Adeno-Associated Virus Vectors Adeno-associated virus (AAV) vectors can efficiently deliver genes to many cell types and produce long- term transgene expression. Although AAV vector genomes often persist without altering a cell's chromosomes, they can also permanently modify the genome through gene targeting or integration at non- homologous sites. In some cases integration is essential for therapeutic or experimental efficacy. In the previous funding period this grant supported our research on AAV-mediated gene targeting and non- homologous integration. Gene targeting refers to the genetic modification of a specific site on a chromosome that shares DNA sequence with the vector. Non-homologous integration refers to the introduction of the vector genome at unrelated, non-specific chromosomal sites. In this proposal we will follow up on our prior results in three areas. In each case we will use AAV vectors to manipulate and probe the mammalian genome, further developing this important vector system. First, we will take advantage of the high frequency and accuracy of AAV-mediated gene targeting to correct several disease-causing mutations in human stem cells. The corrected and uncorrected stem cells produced will serve as ideal cellular models of human disease, and the targeting vectors employed can be used therapeutically when transplantation therapies are developed for patient-derived pluripotent stem cells. Second, using modern sequencing technologies we will determine where AAV-mediated gene targeting and non-homologous vector integration occur on a genome-wide basis. Based on our prior results, this should identify human chromosomal regions prone to breakage, and map recombination frequencies in cultured human cells for the first time. Third, we will follow up on our unexpected observation that AAV vector integration at a specific mouse locus can lead to liver tumors. Given the potential importance of these findings, it is essential that we establish whether this was a unique example unlikely to be encountered in other settings, or a real concern for the clinical use of AAV. This research has significant potential to develop new therapies for genetic disease and establish the risks of AAV vectors.
Relevance The proposed research will improve adeno-associated virus (AAV) vectors, which are increasingly used in research and therapeutic applications. New methods for correcting disease-causing mutations will be developed, which may ultimately be used in cellular therapies. The possible risks of AAV vectors will also be studied, with relevance for their clinical use and the study of liver cancer.
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