Development of genetically modified pig models using CRISPR-CAS9 with precise insertion via BXB1 recombination site for functional and therapeutic studies.

• Grant number: 25/09002-3
• Support Opportunities: Scholarships in Brazil - Post-Doctoral
• Start date: July 01, 2025
• End date: June 30, 2027
• Field of knowledge: Biological Sciences - Genetics - Human and Medical Genetics
• Principal Investigator: Silvano Mario Attilio Raia
• Grantee: Vitor Leão
• Host Institution: Faculdade de Medicina (FM). Universidade de São Paulo (USP). São Paulo , SP, Brazil
• Associated research grant: 21/11872-5 - Science for Development Center in Xenotransplantation, AP.CCD

Abstract

The global shortage of organs for transplantation represents a persistent ethical and logistical challenge for healthcare systems, including in Brazil, where supply still falls short of demand, resulting in long waiting lists and high morbidity and mortality among patients. In this context, xenotransplantation - the use of animal-derived organs, tissues, or cells in humans - emerges as a viable and promising alternative. Pigs stand out as an ideal model for this application, due to their anatomical and physiological similarities to humans, as well as practical advantages such as rapid growth and ease of genetic manipulation.The main obstacle to xenotransplantation is immunological rejection, particularly hyperacute rejection, triggered by the presence of surface antigens such as the ¿-Gal epitope (produced by the enzyme encoded by the *GGTA1* gene) and structures associated with the *CMAH* and *B4GalNT2* enzymes. Simultaneous inactivation of these three genes through gene editing techniques is an effective approach to minimizing the immune response mediated by natural antibodies. Among these technologies, the CRISPR-Cas9 system stands out for its high precision and efficiency in inducing specific mutations.More recently, strategies have been developed to not only eliminate immunogenic antigens via knockout but also to insert human immunomodulatory genes through knock-in approaches, using integrase-based systems such as Bxb1. This approach offers greater control over the gene insertion site, enhancing genomic safety and the stability of the generated cell lines.This project proposes the development of triple-knockout porcine fibroblasts (*GGTA1*, *B4GalNT2*, and *CMAH*), followed by the insertion of human immunomodulatory transgenes using the Bxb1 platform. These cells will serve as donors in somatic cell nuclear transfer cloning protocols, with the aim of generating genetically modified pigs for use in biomedical models and xenotransplantation. Protocols for cell culture, electroporation, genotypic analysis, and protein expression will be standardized to ensure experimental reproducibility and the translational viability of the proposal.The innovation of this project lies in the integration of advanced genetic engineering tools with direct applications in the fields of regenerative medicine, immunology, and transplantation. The generation of porcine models with multiple genetic modifications via CRISPR-Cas9 and targeted insertion via Bxb1 represents a strategic advancement with significant scientific, clinical, and industrial impact.