Swine Somatic Cell Gene Editing Testing Center (Targeted Challenge)

About This Grant

Project Summary The objective of this supplement project is to perform a side-by-side assessment of various gene editing (GE) formulations to identify those most effective as reagents for tissue editing. The project involves the delivery of CRISPR/Cas9 gene editing ribonucleoprotein complexes intravenously to pigs. Fluorescent reporter pigs will be used to detect editing activity and cell transduction efficiency. Successful targeting in cells and tissues by the formulations will be demonstrated by the induced expression of a red fluorescent protein (tdTomato). The work focuses on two efforts 1) ‘Programmable Delivery System for Gene Editing’, which will evaluate organ-specific delivery and 2) ‘Crossing the Blood Brain Barrier (BBB)’, which will evaluate the relative efficiency of reagents to circumvent the BBB to deliver editors to neural tissue. Both project efforts are structured into three parts: 1) In Vitro evaluations of test reagents, 2) In Vivo Toxicity Pilot delivery, and 3) final In Vivo Delivery to complete the animal studies. Importantly, all the reagents will utilize the same guide RNA from a common source that will target the same sites for editing. After each delivery, the pigs will be monitored daily, and blood will be drawn frequently. Inflammatory cytokines will be measured as well as serum chemistry levels and blood CBC and differentials, using the standard toxicology package provided by the University of Missouri Veterinary Medicine Diagnostic Laboratory (VMDL). At the close of the study (4 weeks ±2 days post-delivery), animals will be euthanized, and gross necropsies will be performed by the Testing Center staff. Tissues will be harvested, fixed, embedded, and sectioned for histopathology evaluation and imaging of tdTomato (or other immunostaining, based on project needs). For ‘Programmable Delivery’, three project-defined target tissues, along with the Liver and Thoracic Dorsal Root Ganglion will be evaluated. For ‘Crossing BBB’, two coronal brain slices (A&B) will be hemisected into the left and right hemisphere resulting in four brain areas to be evaluated; additionally, the Liver and Thoracic Dorsal Root Ganglion will be evaluated. Ultimately, these evaluations will include H&E, cell-specific markers in serial sections to determine which cell type(s) were transduced, and high-resolution fluorescent imaging of the most targeted tissues. A detailed summary of the imaging assessments, blood panels, and circulating inflammatory markers will be provided to the targeted challenge board for their rankings. At the end of the study, the results and tissues will be provided to the submitting investigator teams.