Generation and characterization of PET-reporter pigs for noninvasive imaging and cell therapy research

PROJECT SUMMARY Cell therapy is emerging as an important therapeutic tool for a range of intractable and non-druggable diseases. A wide spectrum of therapeutic cells coined as “living drugs” are being developed in recent years for treating a panoply of indications including cancer, hematopoietic diseases, diabetes, liver pathologies, inherited diseases, etc., with few of them in advanced stages of clinical trials, and some (KymriahTM and YescartaTM) approved by FDA for clinical use. A major bottleneck for realizing the full potential of cell therapies is a lack of tools and resources in large animal models such as pigs, which provide the best cost- benefit considerations for safety and efficacy studies, and for better translatability of findings to humans. A clear understanding of cell viability, functionality, and bio-distribution of transplanted cells in the diseased model is paramount for cell therapy research. From a bio-distribution standpoint, precise dosing, delivery of cells to desired sites, tracking the retention of cells in tissues, and clearance over time in vivo is critical. In this regard, novel tools for non- invasive imaging such as positron emission tomography (PET) in large animal models like pigs is essential for improving the accuracy and efficiency of cell therapies in vivo. To address this stated gap, the main goal of this proposal is to develop foundational tools and resources for non-invasively detecting and tracking the implanted cells and tissues in vivo. To achieve this goal, two Specific Aims are proposed. Aim-1 will generate and characterize PET-reporter cells. We plan to generate stable transgenic PET reporter cells by CRISPR/Cas knockin of dual bicistronic PSMA and NIS reporter genes into ROSA26 safe- harbor locus. Functionality of the resulting cells will be validated both in vitro and in vivo. Aim-2 will establish a line of CRE-inducible and CRE-activated dual PET-reporter minipigs: The sequence confirmed, and functionality validated cells will be used for generating live offspring via somatic cell nuclear transfer (SCNT; or cloning). We expect that a successful outcome of this proposal will lay a foundation for non-invasive imaging in pig models- a great need; and will open-up promising new avenues for cell and tissue therapy research.