Zinc finger nucleases ('ZFNs'), TAL effector nucleases '(TALENs'), CRISPR-Cas9 nucleases (?CRISPRs?) and meganuclease/TAL effector fusions ('MegaTALs', which are the focus of this project) are all highly specific nucleases that can generate single- or double-strand breaks at individual genomic loci. Each of these nuclease platforms is being developed for a wide variety of applications, including basic research, industrial and agricultural genome engineering, cellular therapeutics (for example, CAR T-cells), and direct gene therapy. Although CRISPR nucleases are now the system of choice for almost all genome engineering, their utility and performance for therapeutic applications is not a solved problem. For clinical use, nuclease performance is defined by the ease of its packaging and delivery, its activity and specificity in a living cell, and the balance of competing DNA repair outcomes. MegaTAL nucleases display several favorable properties for such purposes, including monomeric structures, small size, high activity and specificity, and unique cleavage mechanisms that produce 3' DNA overhangs. We have generated a large number of engineered MegaTAL nucleases and have described their ex vivo and in vivo performance in primary human cells and transgenic organisms, as summarized in the full text of this project description. While all these four of these platforms are being studied and used for gene therapy, optimization of their properties and behaviors (particularly to drive gene modification via homology-driven correction, rather than gene disruption via mutagenic end-joining) is an important ongoing priority. For any nuclease, the kinetics of DNA binding, cleavage and dissociation (and the corresponding affinity and half-life at each step) can alter the composition, structure and dynamic behavior of the DSB lesion in a manner that might affect each pathway differently. This can lead to significant differences in repair outcomes, as illustrated via our preliminary data. In this renewal application, we propose to leverage our engineered nuclease constructs and recently published results for two Specific Aims: (1) Determine the biophysical and enzymatic parameters of nuclease function that most strongly influence DNA repair outcomes and enhance gene modification via HDR. The overall premise for the first aim is that individual DNA repair pathways and their protein factors are uniquely sensitive to differences in the mechanisms and biophysical behaviors of the enzymes that generate a DSB. (2) Optimize our '2nd generation' of MegaTAL scaffolds (that are reduced in size and that appear to display improved activity and specificity) and corresponding mRNA delivery systems in genome editing directed towards primary hematopoietic stem cells (HSCs). The overall premise for the second aim is that the highly variable (but quite controllable) properties of MegaTALs and their delivery systems are particularly appropriate for assessing the efficiency of genome modification and subsequent persistence of gene edited primary cells, both in culture and upon transplantation and engraftment.

Public Health Relevance

The development of highly specific gene targeting proteins that can induce the site-specific modification of a DNA sequence (and in particular induce the homology-driven modification of an existing sequence) is a critical technology for genome engineering and gene therapy. For human medical therapies, the requirement for absolutely specific gene modification activity with highly predictable modification outcomes in living cells is paramount, but has not been met entirely by existing targeting nucleases. This proposal focuses on one class of gene-targeting nucleases (MegaTALs) that display many highly desirable properties, and examines the mechanistic properties that most clearly effect their performance in gene modification applications.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Project (R01)
Project #
Application #
Study Section
Therapeutic Approaches to Genetic Diseases Study Section (TAG)
Program Officer
Keane-Myers, Andrea
Project Start
Project End
Budget Start
Budget End
Support Year
Fiscal Year
Total Cost
Indirect Cost
Fred Hutchinson Cancer Research Center
United States
Zip Code
Bogdanove, Adam J; Bohm, Andrew; Miller, Jeffrey C et al. (2018) Engineering altered protein-DNA recognition specificity. Nucleic Acids Res 46:4845-4871
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
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
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