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.