A major limitation to effective targeted gene repair is the low efficiency of homology-directed repair in most human cell types. The mechanism of homology-directed repair is now understood at a very sophisticated level of detail, and the goal of this component of the Northwest Genome Engineering Consortium (NGEC) is to harness this mechanistic understanding to develop methods for stimulating HDR at target genes. We describe three specific aims to achieve this goal: (1) We will develop a convenient reporter assay for homology-directed repair, and use it to optimize repair at double-strand breaks, in cell culture models and in primary hematopoietic cells. (2) We will ask if targeted gene repair can be efficiently initiated at nicks; and develop a reporter assay for break-induced genomic instability to establish whether nicks provide a safety advantage over double-strand breaks by diminishing the potential for translocation in therapeutic applications. (3) We will establish how chromatin structure contributes to gene marking and to targeted gene repair, and ask specifically if chromatin structure can be manipulated to increase the efficiency of gene repair. These planned experiments complement and mesh with other components of the NGEC to achieve the long-term goal of establishing methods for effective gene therapy of monogenic disorders of hematopoietic cells.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Linked Research project Grant (RL1)
Project #
5RL1GM084434-03
Application #
7646269
Study Section
Special Emphasis Panel (ZRR1-SRC (99))
Project Start
2007-09-17
Project End
2012-06-30
Budget Start
2009-07-01
Budget End
2010-06-30
Support Year
3
Fiscal Year
2009
Total Cost
$382,590
Indirect Cost
Name
University of Washington
Department
Microbiology/Immun/Virology
Type
Schools of Medicine
DUNS #
605799469
City
Seattle
State
WA
Country
United States
Zip Code
98195
Le, Quy; Maizels, Nancy (2015) Cell Cycle Regulates Nuclear Stability of AID and Determines the Cellular Response to AID. PLoS Genet 11:e1005411
Kuhar, Ryan; Gwiazda, Kamila S; Humbert, Olivier et al. (2014) Novel fluorescent genome editing reporters for monitoring DNA repair pathway utilization at endonuclease-induced breaks. Nucleic Acids Res 42:e4
Davis, Luther; Maizels, Nancy (2014) Homology-directed repair of DNA nicks via pathways distinct from canonical double-strand break repair. Proc Natl Acad Sci U S A 111:E924-32
Maizels, Nancy (2013) Genome engineering with Cre-loxP. J Immunol 191:5-6
Humbert, Olivier; Davis, Luther; Maizels, Nancy (2012) Targeted gene therapies: tools, applications, optimization. Crit Rev Biochem Mol Biol 47:264-81
Humbert, Olivier; Maizels, Nancy (2012) Epigenetic modification of the repair donor regulates targeted gene correction. Mol Ther Nucleic Acids 1:e49
Davis, Luther; Maizels, Nancy (2011) DNA nicks promote efficient and safe targeted gene correction. PLoS One 6:e23981
Ordinario, Ellen C; Yabuki, Munehisa; Handa, Priya et al. (2009) RAD51 paralogs promote homology-directed repair at diversifying immunoglobulin V regions. BMC Mol Biol 10:98
McConnell Smith, Audrey; Takeuchi, Ryo; Pellenz, Stefan et al. (2009) Generation of a nicking enzyme that stimulates site-specific gene conversion from the I-AniI LAGLIDADG homing endonuclease. Proc Natl Acad Sci U S A 106:5099-104
Cummings, W Jason; Bednarski, David W; Maizels, Nancy (2008) Genetic variation stimulated by epigenetic modification. PLoS One 3:e4075