Enhancing CRISPR-based Therapeutic Interventions Via a Targeted Platform Tool
Project Summary
The approach described in this proposal anticipates the needs of translational drug
development by designing and manufacturing a broadly applicable platform tool that eases
CRISPR-based therapeutic prioritization: modular, targeted, cell-specific delivery of Cas9.
Pursuit of gene therapies for any cell-based disorder becomes more efficient through the tool's
versatility during in vitro discovery phases, continuity through in vivo preclinical pharm-tox
phases, and potential for human clinical administration without compromising accuracy in
gene-altering outcomes.
The platform approach described in this proposal incorporates the modular flexibility of
streptavidin-biotin interactions to provide a tool that allows drug discovery researchers to
efficiently swap-out a variety of antibodies to prioritize different specific cell targets, eliminating
the need to create a unique CRISPR toolset for each cell-type of interest. By leveraging the
use of antibodies to target cell-based diseases, as in cancer intervention, the current proposal
intends to harness the same specific binding properties of antibodies to deliver CRISPR/Cas9
gene-related therapies. Enabling researchers to target specific cells within a larger
heterogeneous cell population either in vivo or in vitro anticipates downstream drug candidate
validation needs.
Phase I of this proposal describes an experimental design to validate the efficiency of
CRISPR gene editing when incorporating cell-specific targeted delivery of the Cas9
component. Through separation at the cellular level of the targeted delivery of the gene-
editing instrument (Cas9) from the delivery of nucleus-targeted genetic information, therapeutic
potential is enhanced by this safety mechanism, preventing off-target gene altering in
undesired cell populations. Drawing on this “molecular surgery” technology to specifically
target cell-populations based on receptor expression, a universal platform tool comprised of
Cas9 attached to Streptavidin will be developed and examined for enhancement of CRISPR-
based therapeutic identification. The development process will include: 1) Recombinant
production of an enhanced specificity Cas9 with bespoke conjugation sites for crosslinking, 2)
Conjugation and purification of a conjugate comprising streptavidin and recombinant Cas9,
and 3) Confirmation of selective specific delivery of Cas9 and retained editing ability to delete a
gene within stem cell populations. This project will validate the cell-specific targeting approach
to deliver Cas9 via in vitro procedures and testing with a keen eye to the needs of in vivo
testing planned for Phase II.