Journal
MOLECULAR THERAPY
Volume 18, Issue 6, Pages 1103-1110Publisher
CELL PRESS
DOI: 10.1038/mt.2010.57
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Funding
- National Institutes of Health (NIH) [PN2EY018244, R01 HL079295, K08 H1070268]
- Burroughs Wellcome Fund
- State of Texas through the University of Texas Southwestern Medical Center
- UTSW Medical Scientist
- National Institutes of Healthgrant at the University of Texas Southwestern Medical Center
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Zinc Finger nucleases (ZFNs) have been used to create precise genome modifications at frequencies that might be therapeutically useful in gene therapy. We created a mouse model of a generic recessive genetic disease to establish a preclinical system to develop the use of ZFN-mediated gene correction for gene therapy. We knocked a mutated GFP gene into the ROSA26 locus in murine embryonic stem (ES) cells and used these cells to create a transgenic mouse. We used ZFNs to determine the frequency of gene correction by gene targeting in different primary cells from this model. We achieved targeting frequencies from 0.17 to 6% in different cell types, including primary fibroblasts and astrocytes. We demonstrate that ex vivo gene-corrected fibroblasts can be transplanted back into a mouse where they retained the corrected phenotype. In addition, we achieved targeting frequencies of over 1% in ES cells, and the targeted ES cells retained the ability to differentiate into cell types from all three germline lineages. In summary, potentially therapeutically relevant frequencies of ZFN-mediated gene targeting can be achieved in a variety of primary cells and these cells can then be transplanted back into a recipient.
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