Using zinc finger nucleases to manipulate the mammalian genome





Matthew H. Porteus

Department of Pediatrics and Department of Biochemistry, University of Texas Southwestern Medical Center, 5323 Harry Hines Blvd, Dallas, Texas 75390, USA



Gene targeting is the replacement of an endogenous segment of DNA with an exogenous segment by homologous recombination. Through gene targeting both small and large sequence changes can be introduced into the genome and it is the most precise method of genomic manipulation. It has been a cornerstone of research in yeast and murine embryonic stem cells. Because of its precision, gene targeting could be an ideal approach to treating monogenic diseases. In mammalian somatic cells, however, gene targeting only occurs spontaneously in one a million cells -- a frequency that makes it impractical for either experimental or therapeutic purposes. A critical development was the discovery that a DNA double-strand break (DSB) in the target gene could stimulate the process by several-thousand fold -- a finding that suggested that if one could develop a reagent to create target gene specific DSBs one might be able to increase the rate of gene targeting to levels that would be practically useful. Zinc finger nucleases (ZFNs) are artificial proteins that fuse the DNA binding from a zinc finger to a non-specific nuclease domain. Using a step-wise approach, we have shown that model ZFNs can stimulate targeting in a reporter system, that designed ZFNs can stimulate targeting in a reporter system, and that designed ZFNs can stimulate targeting in endogenous genes. When we designed ZFNs to target the IL2RG gene, the gene mutated in the most common form of severe combined immunodeficiency (SCID), we were able to achieve targeting frequencies of 20% in cell lines and 5% in primary human T-cells. These rates of targeting are of a level which are practically useful for both experimental and therapeutic purposes. For therapeutic purposes, however, several outstanding issues need to be resolvedincluding the need to minimize "off-target" effects, developing efficient methods to develop highly specific zinc fingers, and finding the optimal delivery into stem cells.




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