LAGLIDADG homing endonucleases ('LHEs', also termed 'meganucleases'), zinc finger nucleases ('ZFNs') TAL effector nucleases '(TALENs') and CRISPR nucleases are DNA cleavage systems that are used for genome engineering and corrective gene therapy. These systems display specificity profiles that correspond to varying amounts of off-target activity in the human genome. To address this issue, we have (1) developed methods for LHE engineering that is appropriate for academic laboratories (PNAS 2011); (2) determined the structure and recognition mechanism of a TAL effector (Science 2012); and (3) created and characterized a hyperspecific gene targeting scaffold termed a 'MegaTAL' endonuclease, that exploits the combined properties and mechanisms of TAL effectors and meganucleases.
Aim 1 : Create engineered MegaTALs to target two individual human disease-associated loci, then correlate their in vitro properties to their activities in transfected human cell lines. We have generated meganucleases that nick or cleave DNA target sites associated with two significant human genetic disorders (hemoglobinopathies and cystic fibrosis). The first of these therapeutic targets requires ex vivo disruption of a silencing regionin the -globin locus in patient-derived hematopoietic cell lines, whereas the second application requires in vivo targeted gene correction in the lung epithelium. We hypothesize that the MegaTALs will display both increased cleavage activity (by localizing the endonuclease to the target) and exceptional specificity for that same DNA sequence. We will test the hypothesis by characterizing: (1) the effect of the TAL anchor on gene conversion levels; and (2) the effect of the TAL anchor on genome-wide cleavage profiles and off-target activities.
Aim 2 : Augment the MegaTAL scaffold with tailored nucleolytic activities and molecular recruitment domains that can reduce undesired repair outcomes and/or enhance gene conversion activity. We have developed strategies both to diversify the nucleolytic activity of the LHE (to generate site specific nickases and cleavases for the same targets) and to recruit corrective DNA templates and/or recombination machinery to the target by the meganuclease. We hypothesize that these constructs can be exploited to further reduce or eliminate undesirable off-target activity, mutagenesis and toxicity, and will test that hypothesis using a panel of cellular assays for DNA repair, fidelity and viability as described in the proposal.
The development of highly specific gene targeting proteins that can induce the site-specific modification of a DNA sequence is a critical technology for genome engineering and (in particular) for corrective gene therapy. For human medical therapies, the requirement for absolutely specific, single-site activity and gene modification is paramount, and has not yet been met by existing targeting reagents such as zinc finger nucleases. This proposal directly addresses that need, by creating a new class of gene targeting reagents.
|Bogdanove, Adam J; Bohm, Andrew; Miller, Jeffrey C et al. (2018) Engineering altered protein-DNA recognition specificity. Nucleic Acids Res 46:4845-4871|
|Shen, Betty W; Heiter, Daniel F; Lunnen, Keith D et al. (2017) DNA recognition by the SwaI restriction endonuclease involves unusual distortion of an 8 base pair A:T-rich target. Nucleic Acids Res 45:1516-1528|
|Rinaldi, Fabio C; Doyle, Lindsey A; Stoddard, Barry L et al. (2017) The effect of increasing numbers of repeats on TAL effector DNA binding specificity. Nucleic Acids Res 45:6960-6970|
|Werther, Rachel; Hallinan, Jazmine P; Lambert, Abigail R et al. (2017) Crystallographic analyses illustrate significant plasticity and efficient recoding of meganuclease target specificity. Nucleic Acids Res 45:8621-8634|
|Niyonzima, Nixon; Lambert, Abigail R; Werther, Rachel et al. (2017) Tuning DNA binding affinity and cleavage specificity of an engineered gene-targeting nuclease via surface display, flow cytometry and cellular analyses. Protein Eng Des Sel 30:503-522|
|Moffett, H F; Coon, M E; Radtke, S et al. (2017) Hit-and-run programming of therapeutic cytoreagents using mRNA nanocarriers. Nat Commun 8:389|
|Lambert, Abigail R; Hallinan, Jazmine P; Shen, Betty W et al. (2016) Indirect DNA Sequence Recognition and Its Impact on Nuclease Cleavage Activity. Structure 24:862-73|
|Romano Ibarra, Guillermo S; Paul, Biswajit; Sather, Blythe D et al. (2016) Efficient Modification of the CCR5 Locus in Primary Human T Cells With megaTAL Nuclease Establishes HIV-1 Resistance. Mol Ther Nucleic Acids 5:e352|
|Shen, Betty W; Lambert, Abigail; Walker, Bradley C et al. (2016) The Structural Basis of Asymmetry in DNA Binding and Cleavage as Exhibited by the I-SmaMI LAGLIDADG Meganuclease. J Mol Biol 428:206-220|
|Takeuchi, Ryo; Choi, Michael; Stoddard, Barry L (2014) Redesign of extensive protein-DNA interfaces of meganucleases using iterative cycles of in vitro compartmentalization. Proc Natl Acad Sci U S A 111:4061-6|
Showing the most recent 10 out of 11 publications