DEVELOPING ZFNs AS MOLECULAR TOOLS FOR TARGETED INTEGRATION A sought after goal of molecular biologists has been the ability to manipulate or modify plant and mammalian genomes including the human genome at specific sites. Gene targeting - the process of replacing a gene by homologous recombination (HR) - uses an extra-chromosomal fragment of template DNA and invokes the cell's own repair machinery for gene conversion. Gene targeting is not a very efficient process in mammalian cells; about only 1 in a million treated cells undergo the desired gene modification. Zinc finger nucleases (ZFNs) that combine the non-specific cleavage domain (N) of Fokl endonuclease with zinc finger proteins (ZFPs) offer a general way to deliver site-specific DSB to the genome, and stimulate local HR to repair the DSB. Several labs have shown that ZFNs find and cleave their chromosomal target in cells and stimulate local HR to repair the DSB. In this proposal, we address 3 barriers to wider implementation of ZFN-mediated gene targeting in Genetic Medicine. First, existing strategies for selecting ZFPs with high affinity and strict sequence-specificity are laborious and cumbersome. We have developed bacterial one-hybrid, single-reporter systems to rapidly select and refine sequence-specificity of ZFNs, which we propose to further test. Second, even though our 3-finger ZFNs cleave specific sites in vivo, they may be toxic to cells due to cleavage at secondary (non-targeted) sites. Imparting greater sequence-specificity to the ZFNs, we hypothesize, will make them less toxic. We propose to examine the efficacy of ZFN-mediated gene targeting in cells by comparing pairs of 3- and 4-finger ZFNs, recognizing 18 bp and 24 bp DNA sequence, respectively. Third, precisely targeted site-specific modification of 2 important human cell types, namely primary CD34+ haematopoietic stem-progenitor cells (HSPCs) and embryonic stem cells (HESCs), cannot be done effectively by routine technologies. This limits laboratory research in human cells and potential translation to clinical therapies. We propose to test the efficacy and toxicity of ZFN-mediated gene targeting in these key cell types. We have chosen the chemokine (C-C motif) receptor 5 (CCR5) locus of the human genome as a model locus for ZFN-mediated targeted disruption in primary human HSPCs and HESCs. ? ? ?

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
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Gene and Drug Delivery Systems Study Section (GDD)
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Portnoy, Matthew
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Johns Hopkins University
Public Health & Prev Medicine
Schools of Public Health
United States
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Chandrasegaran, Srinivasan (2017) Recent advances in the use of ZFN-mediated gene editing for human gene therapy. Cell Gene Ther Insights 3:33-41
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