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.
Public Health Relevance Statement: Enhancing CRISPR-based Therapeutic Interventions Via a Targeted Platform Tool Narrative The search for effective therapeutics often begins through examination of cell-specific function in diseases and disorders; drug candidates frequently target specific cells to deliver a payload that can cure or treat the problem (cancer, Alzheimer's Disease, chronic pain, Parkinson's Disease, to name a few). The drug development platform approach described in this proposal provides a broadly applicable tool that enhances the efficiency of CRISPR-based therapeutic development with targeted, cell-specific delivery of Cas9. Doing so makes any cell-based disorder a candidate for gene-based clinical intervention through versatility in administration without compromising accuracy in outcomes.
Project Terms: Adverse effects; Alzheimer's Disease; Amino Acid Sequence; Antibodies; Antibody-drug conjugates; Applications Grants; base; Basic Science; Binding; Biotin; Cancer Intervention; cancer therapy; Candidate Disease Gene; candidate validation; Cell Nucleus; cell type; Cells; chronic pain; Clinic; Clinical; clinical application; Clustered Regularly Interspaced Short Palindromic Repeats; CRISPR/Cas technology; crosslink; design; Development; Disease; DNA Sequence Alteration; drug candidate; drug development; drug discovery; Epitopes; Event; experience; Experimental Designs; Explosion; extracellular; Eye; flexibility; Gene Targeting; gene therapy; Genes; genetic information; Genome; genome editing; Genomics; Hereditary Disease; HIV; Human; In Vitro; in vitro testing; in vivo; instrument; interest; Intervention; Label; Ligands; Malignant Neoplasms; Methods; Molecular; Molecular Weight; Muscular Dystrophies; Names; Operative Surgical Procedures; Outcome; Parkinson Disease; Patients; Phase; Population; population based; Population Heterogeneity; pre-clinical; prevent; Procedures; Process; Production; Property; prospective; Proteins; receptor expression; Recombinants; Regenerative Medicine; relating to nervous system; Reporting; Research Personnel; Retinitis; Ribosomes; Safety; Site; Specificity; Staging; standard of care; stem cell population; stem cell therapy; Stem cells; Streptavidin; System; targeted delivery; Targeted Toxins; Technology; Testing; Therapeutic; therapeutic development; Therapeutic Intervention; tool
