Revealing the DNA Cleavage Specificities of Therapeutically Relevant Endonucleases Through In Vitro Selection

Sequence-specific endonucleases, such as homing endonucleases and zinc finger nucleases, are potentially safer alternatives to virus-mediated strategies for genome engineering. While retroviruses are effective at inserting genes into genomes, they do so randomly, and their use has resulted in malignancy in at least one clinical trial. In contrast to randomly integrating viruses, sequence-specific endonucleases create double-strand breaks in targeted locations in the genome. The double-strand breaks can be resolved through homologous recombination with an exogenous donor, effecting gene replacement, or through the error-prone non-homologous end joining pathway, causing targeted gene knockout. Since off-target cleavage is a possibility, cleavage specificity studies on endonucleases designed for use in gene therapy applications are necessary to ensure that off-target cleavage events do not result in malignancy or other deleterious outcomes. In-depth studies of endonuclease target site cleavage can also provide insights that can be used to improve specificity.;In Chapter One, current methods to both engineer and profile three types of sequence-specific endonucleases (homing endonucleases, zinc finger nucleases, and engineered TAL nucleases) are reviewed. Chapter Two describes the application of a previously described in vitro selection to profile the specificity of the 1-SceI homing endonuclease, as well as efforts to design a new in vitro selection for endonuclease target sites. Chapter Three describes the application of the new in vitro selection in, to our knowledge, the first comprehensive study of zinc finger nuclease (ZFN) cleavage specificity. In Chapter Three, we profile the VEGF-A promoter-targeting VF2468 ZFN and the CCRS-224 ZFN, which targets the CCR5 gene and is currently in one Phase 1/2 and two Phase 1 clinical trials for the treatment of HIV. We identify nine off-target sites for CCR5-224, including one in the promoter of BTBD10, a gene that has previously been associated with malignancy. In addition, we motivate a model for ZFN specificity in which excess binding energy enables off-target cleavage events. In Chapter Four, we report improvements made on the in vitro selection used in Chapter Three and discuss the future application of the in vitro selections described in this thesis to study engineered TAL nucleases.