Development of porcine stem cell-based technologies to improve the efficiency, reliability, and reproducibility of animal models

About This Grant

PROJECT SUMMARY Rodent models have been foundational for biomedical discovery for decades. Yet the limitations of mice as a “universal” biomedical model are becoming increasingly apparent. Large animal models like minipigs offer a transformative opportunity to bridge this gap. Among the available minipig breeds, the naturally obesogenic Ossabaw minipig is increasingly becoming a model of choice for modeling the metabolic and chronic disease burdens that account for over 90% of U.S. healthcare costs. However, the generation of genetically engineered minipigs (GEMPs) remains inefficient and poorly scalable, limiting their utility in disease modeling and translational research. This proposal aims to overcome these limitations by establishing a precision genetic engineering (GE) and chimera generation platform using porcine induced pluripotent stem cells (piPSCs). The piPSC confer many advantages for GE efforts including the lack of senescence and maintenance over multiple generations, ease for genetic modification, potential for serial and multiplex editing, high throughput mutational screens, and contribution to soma and germline in the chimeric offspring. These attributes are eminently desirable for this animal resource. Our central hypothesis is that chimera-competent piPSC are a superior platform for programmable, high-efficiency GE and for generating functional chimeras via embryo complementation. To test this hypothesis, we propose the following two Specific Aims: Aim 1 will develop and validate a high-efficiency, site-specific genome engineering platform in Ossabaw piPSC. We will develop a high-fidelity piPSC based serine recombinases (Bxb1, PA01, KP03) platform for robust and modular GE. By targeting a universal landing pad to the pROSA26 safe harbor locus via CRISPR/Cas9, we will enable site-specific, scalable, and reproducible transgene integration, including large constructs like BACs. As a proof-of-concept, we will generate two novel models: (i) Cre-inducible dual fluorescent-PET reporter pigs for in vivo tracking and imaging, and (ii) a humanized CETP-transgenic minipig that addresses a key interspecies gap in lipoprotein metabolism and cardiovascular disease research. Aim 2 will enable somatic and germline chimerism using piPSC in lineage-deficient embryos. By modulating pluripotency state, injection parameters, and using lineage-deficient host embryos (e.g., lacking IGFR1, HHEX, PAX4, or NANOS3), we will demonstrate donor cell integration, niche occupancy, and rescue of organogenesis or gametogenesis. Donor contribution will be validated using advanced tools such as spatial transcriptomics, immunohistochemistry, and ddPCR, and functionality validated by phenotyping the founder animals. In summary, the proposal will establish and validate a high-efficiency, precision pipeline for producing GEMPs and functional chimeras. The stem cell tools and resources developed in this proposal will firmly establish Ossabaw minipigs as one of the models of choice for preclinical research.