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 In recently published work and in unpublished data we have demonstrated that reducing certain NHEJ factors does indeed increase the frequency of rAAV-mediated gene targeting.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.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM088351-03
Application #
8233524
Study Section
Special Emphasis Panel (ZRG1-TAG-M (01))
Program Officer
Janes, Daniel E
Project Start
2010-03-15
Project End
2014-02-28
Budget Start
2012-03-01
Budget End
2013-02-28
Support Year
3
Fiscal Year
2012
Total Cost
$283,687
Indirect Cost
$90,637
Name
University of Minnesota Twin Cities
Department
Biochemistry
Type
Schools of Arts and Sciences
DUNS #
555917996
City
Minneapolis
State
MN
Country
United States
Zip Code
55455
Harvey, Adam; Mielke, Nicholas; Grimstead, Julia W et al. (2018) PARP1 is required for preserving telomeric integrity but is dispensable for A-NHEJ. Oncotarget 9:34821-34837
Baird, Duncan M; Hendrickson, Eric A (2018) Telomeres and Chromosomal Translocations : There's a Ligase at the End of the Translocation. Adv Exp Med Biol 1044:89-112
Thompson, Elizabeth L; Yeo, Jung E; Lee, Eun-A et al. (2017) FANCI and FANCD2 have common as well as independent functions during the cellular replication stress response. Nucleic Acids Res 45:11837-11857
Kan, Yinan; Batada, Nizar N; Hendrickson, Eric A (2017) Human somatic cells deficient for RAD52 are impaired for viral integration and compromised for most aspects of homology-directed repair. DNA Repair (Amst) 55:64-75
Kan, Yinan; Ruis, Brian; Takasugi, Taylor et al. (2017) Mechanisms of precise genome editing using oligonucleotide donors. Genome Res 27:1099-1111
Alotaibi, Moureq; Sharma, Khushboo; Saleh, Tareq et al. (2016) Radiosensitization by PARP Inhibition in DNA Repair Proficient and Deficient Tumor Cells: Proliferative Recovery in Senescent Cells. Radiat Res 185:229-45
Neal, Jessica A; Xu, Yao; Abe, Masumi et al. (2016) Restoration of ATM Expression in DNA-PKcs-Deficient Cells Inhibits Signal End Joining. J Immunol 196:3032-42
Liddiard, Kate; Ruis, Brian; Takasugi, Taylor et al. (2016) Sister chromatid telomere fusions, but not NHEJ-mediated inter-chromosomal telomere fusions, occur independently of DNA ligases 3 and 4. Genome Res 26:588-600
Castella, Maria; Jacquemont, Celine; Thompson, Elizabeth L et al. (2015) FANCI Regulates Recruitment of the FA Core Complex at Sites of DNA Damage Independently of FANCD2. PLoS Genet 11:e1005563
Hendrickson, Eric A; Baird, Duncan M (2015) Alternative end joining, clonal evolution, and escape from a telomere-driven crisis. Mol Cell Oncol 2:e975623

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