The long term goal of the proposed research is to engineer new, highly sequence-specific DMA binding proteins with the ability to target and cleave specific human genes.
Our aim i s to generate and use new Gene-Specific Reagents (GSR's) to promote the efficient recombinational repair or ablation of specific genes in living cells. Novel GSR's will be generated from preexisting homing endonuclease proteins of the LAGLIDADG family. These homing endonucleases catalyze the site-specific lateral transfer of parasitic DNA elements in all Kingdoms of life, and already possess many desirable properties for GSR engineering. For . example, LAGLIDADG homing endonucleases have long DNA target sites of 18-24 bp, a high degree of DNA sequence specificity, and tight coupling of DNA site recognition to DNA strand cleavage. This last property allows homing endonucleases to precisely target DNA strand cleavage to single phosphodiester bonds in complex genomes with little or no 'collateral damage'as a result of off-target or spurious cleavage events. In the proposed research we will generate LAGLIDADG homing endonuclease variants that target 9 human genes and their canine or murine counterparts. These gene targets were chosen for their role in heritable human hematopoietic disease;in heritable immune deficiency syndromes;or in tyrosinemia type I, one of the most common heritable inborn errors of metabolism. Our experimental Aims are:
Aim 1 : Generate optimized LAGLIDADG homing endonuclease proteins for mammalian genome engineering Aim 2: Develop gene-specific targeting variants of LAGLIDADG homing endonucleases directed against human disease gene targets Aim 3: Determine ability of engineered, gene-specific LAGLIDADG variant proteins to catalyze recombinational gene repair or gene inactivation in vivo.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Linked Research project Grant (RL1)
Project #
5RL1CA133831-04
Application #
7869386
Study Section
Special Emphasis Panel (ZRR1-SRC (99))
Program Officer
Knowlton, John R
Project Start
2007-09-28
Project End
2012-06-30
Budget Start
2010-07-01
Budget End
2011-06-30
Support Year
4
Fiscal Year
2010
Total Cost
$323,062
Indirect Cost
Name
University of Washington
Department
Pathology
Type
Schools of Medicine
DUNS #
605799469
City
Seattle
State
WA
Country
United States
Zip Code
98195
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