We propose to study the impact of loss-of-function mutations of human DNA DSB (double-strand break) repair genes on rAAV (recombinant adeno-associated virus) vector integration. rAAV vectors have two powerful applications and they are in wide use in gene therapy studies. First, they can be used to deliver transgenes to cells. This aspect of rAAV, however, is plagued by the same insertional mutagenesis problems associated with other vectors. A second application for rAAV, however, is the recombination of the vector genome with its cognate homologous chromosomal sequences (aka, gene targeting) using HR (homologous recombination), which is a highly desirable outcome for basic researchers in need of tools to modify the genome and of great utility to gene therapists. However, because NHEJ (non-homologous end joining - the major pathway for DNA DSB repair - predominates in human cells over HR, random rAAV integration events generally occur much more frequently than correct gene targeting events. Therefore, therapeutic inactivation of the NHEJ pathway should augment rAAV-mediated gene targeting and - reciprocally - inactivation of the HR pathway should hinder rAAV gene targeting. In recently published work and in unpublished data we have demonstrated that reducing certain NHEJ factors does indeed increase the frequency of rAAV-mediated gene targeting. In this application we propose to extend this work to other NHEJ factors and to HR-regulated integrations. Moreover, we propose models - and experiments designed to test them - that should illuminate the mechanism that rAAV uses for gene targeting.

Public Health Relevance

Recombinant adeno-associated virus is one of the most promising vectors for gene therapy. We have discovered a way to enhance the utility of this virus by modifying the status of DNA repair genes within human somatic cells. The reduction in expression of certain DNA repair genes elevates the frequencies with which recombinant adeno- associated virus performs gene targeting. The enhanced ability to perform gene therapy is clearly relevant to the mission of NIH.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Project (R01)
Project #
Application #
Study Section
Special Emphasis Panel (ZRG1-TAG-M (01))
Program Officer
Janes, Daniel E
Project Start
Project End
Budget Start
Budget End
Support Year
Fiscal Year
Total Cost
Indirect Cost
University of Minnesota Twin Cities
Schools of Arts and Sciences
United States
Zip Code
Jones, Rhiannon E; Oh, Sehyun; Grimstead, Julia W et al. (2014) Escape from telomere-driven crisis is DNA ligase III dependent. Cell Rep 8:1063-76
Oh, Sehyun; Harvey, Adam; Zimbric, Jacob et al. (2014) DNA ligase III and DNA ligase IV carry out genetically distinct forms of end joining in human somatic cells. DNA Repair (Amst) 21:97-110
Karanja, Kenneth K; Lee, Eu Han; Hendrickson, Eric A et al. (2014) Preventing over-resection by DNA2 helicase/nuclease suppresses repair defects in Fanconi anemia cells. Cell Cycle 13:1540-50
Fattah, Farjana Jahan; Kweon, Junghun; Wang, Yongbao et al. (2014) A role for XLF in DNA repair and recombination in human somatic cells. DNA Repair (Amst) 15:39-53
Kan, Yinan; Ruis, Brian; Lin, Sherry et al. (2014) The mechanism of gene targeting in human somatic cells. PLoS Genet 10:e1004251
Yeo, Jung Eun; Lee, Eu Han; Hendrickson, Eric A et al. (2014) CtIP mediates replication fork recovery in a FANCD2-regulated manner. Hum Mol Genet 23:3695-705
Smith, Stephanie; Fox, Jennifer; Mejia, Marco et al. (2014) Histone deacetylase inhibitors selectively target homology dependent DNA repair defective cells and elevate non-homologous endjoining activity. PLoS One 9:e87203
Waters, Crystal A; Strande, Natasha T; Pryor, John M et al. (2014) The fidelity of the ligation step determines how ends are resolved during nonhomologous end joining. Nat Commun 5:4286
Ghezraoui, Hind; Piganeau, Marion; Renouf, Benjamin et al. (2014) Chromosomal translocations in human cells are generated by canonical nonhomologous end-joining. Mol Cell 55:829-42
Oh, Sehyun; Wang, Yongbao; Zimbric, Jacob et al. (2013) Human LIGIV is synthetically lethal with the loss of Rad54B-dependent recombination and is required for certain chromosome fusion events induced by telomere dysfunction. Nucleic Acids Res 41:1734-49

Showing the most recent 10 out of 11 publications