Establishment of porcine transgenic cell line with potential for immune escape forxenotransplantation in humans

• Grant number: 20/11523-8
• Support Opportunities: Scholarships in Brazil - Post-Doctoral
• Start date: November 01, 2020
• End date: July 31, 2022
• Field of knowledge: Biological Sciences - Genetics - Human and Medical Genetics
• Principal Investigator: Mayana Zatz
• Grantee: Lylyan Fragoso Pimentel
• Host Institution: Instituto de Biociências (IB). Universidade de São Paulo (USP). São Paulo , SP, Brazil
• Company: Universidade de São Paulo (USP). Faculdade de Medicina (FM)
• CNAE: Atividades de atenção à saúde humana não especificadas anteriormente
• CNAE: Atividades de atenção à saúde humana não especificadas anteriormente
• Associated research grant: 18/14275-5 - National production of genetically modified pigs for organ xenotransplantation in humans, AP.PITE

Abstract

Xenotransplantation has achieved great progress over the last years, especiallyfollowing the advances of genetic engineering technologies. In this context, the use of pigs as an organ source has drawn attention due to the reproductive advantages and the anatomical and physiological similarities with primates. However, despite the benefits provided by the use of a pig model, organ transplantation to humans faces complex immunological barriers. These obstacles include of cascades of innate and adaptive immune response, coagulatory and inflammatory dysfcuntions that, at different levels, triggers the recruitment of natural antibodies, activation of T lymphocytes, NK cells and macrophages. Among the factors involved in the organ rejection pathways, some proteins stands out as key players such as PDL1 and CD47, involved in the cellular immune response, CD46, that regulates the complement system, HO-1 and TBM required in inflammation and clotting events. In vitro and in vivo tests demonstrated that the transgenic expression of these human genes in pigs conferred less immunogenicity and agreater immunological protection to transplanted organs. With the use of Neon electroporation system, the present work will transfect transposon piggyBac vectors containing the ORFs of the aforementioned human transgenes into primary porcine aortic endothelial cells under the background triple knockout for the GGTA1, BGalNT2 and CMAH genes. The confirmation of the insertions in the porcine genome as well as expression analysis and selection of edited cells will be carried out by genome sequencing, flow cytometry, RT-qPCR and Western Blotting. Thus, it is expected to establish a cell line with characteristics of a potential model capable of overcoming the immune barriers against xenotransplantation. (AU)