Enhancing CRISPR-based Therapeutic Interventions Via a Targeted Platform Tool

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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.