Gene targeting is a technique used to precisely manipulate a specific chromosomal sequences in living cells. It is used to study how genes function and to correct mutations that can cause disease. Since 1999, this grant has funded our research on the use of adeno-associated virus (AAV) vectors for gene targeting. In that time we have characterized several aspects of the AAV gene targeting technology, which involves a single- stranded linear DNA vector genome that is efficiently delivered to the cell nucleus. Unique aspects of the AAV technology include high targeting frequencies in normal mammalian cells without the use of site-specific nucleases to stimulate recombination, versatility in the typs of sequence changes that can be introduced, high accuracy, and efficient gene targeting in living animals. Because of these advantages, many labs have now used AAV vectors in their gene targeting experiments, and it is being developed for clinical applications as well. In the lat funding period we studied targeting on a genome-wide level for the first time, and we engineered the chromosomes of human pluripotent stem cells (ESCs and iPSCs), which are especially important for understanding and treating human diseases. Here we propose to extend these studies in three ways. First, we will use strand-specific vectors to understand how the single-stranded vector genome recombines with the chromosome, and how this relates to the transient single-stranded regions observed during chromosome replication and transcription. Second, we will identify and then express small inhibitory shRNAs during the gene targeting process both to decrease random integration and to increase homologous recombination. Third, we will combine AAV targeting vectors with the CRIPSR site-specific nuclease system to introduce site-specific nicks that should increase targeting in vivo with minimal genotoxicity. These experiments will increase our understanding of the gene targeting mechanism, maximize the targeting frequencies that can be obtained by AAV vectors, and develop a robust technology for manipulating genes in human stem cells and in vivo applications.

Public Health Relevance

The proposed research will improve a fundamental technology used to manipulate human genes. Advances in gene targeting methods have a significant impact on the types of genetic experiments that can be performed, and on our ability to correct mutations, with potential impact for many types of human diseases. We focus on improving the gene targeting technology in human pluripotent stem cells, which can be used as cellular disease models and developed for cellular therapies, and on correcting genes in the cells of living organisms.

National Institute of Health (NIH)
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Research Project (R01)
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Gene and Drug Delivery Systems Study Section (GDD)
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Leschek, Ellen W
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University of Washington
Internal Medicine/Medicine
Schools of Medicine
United States
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Hiramoto, Takafumi; Li, Li B; Funk, Sarah E et al. (2018) Nuclease-free Adeno-Associated Virus-Mediated Il2rg Gene Editing in X-SCID Mice. Mol Ther 26:1255-1265
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