Development and Characterization of a New Universal Donor iPSC Cell Line

• Grant number: 24/10799-0
• Support Opportunities: Scholarships in Brazil - Master
• Start date: February 01, 2025
• End date: January 31, 2026
• Field of knowledge: Biological Sciences - Genetics - Human and Medical Genetics
• Principal Investigator: Ernesto da Silveira Goulart Guimarães
• Grantee: Gustavo Chagas Santos
• Host Institution: Instituto de Biociências (IB). Universidade de São Paulo (USP). São Paulo , SP, Brazil

Abstract

Induced pluripotent stem cells (iPSCs) are reprogrammed cell lines derived in vitro from fibroblasts or erythroblasts which, similar to embryonic stem cells (ESCs), can differentiate into all tissues of an organism. Due to their high proliferative potential and permissiveness to gene editing and clonal selection, iPSCs are highly applicable for cellular therapies in humans. Immunological rejection is a major challenge in the field of cellular therapy, as cells of the immune system identify transplanted cells as foreign and eliminate them. Human leukocyte antigen class I (HLA-I) is primarily responsible for allogeneic rejection, presenting peptide antigens to CD8+ T cells and initiating cytotoxic immune responses. Beta-2 microglobulin (B2M) stabilizes HLA class I molecules on the cell surface. To prevent CD8+ T cell-mediated immune rejection, it is possible to: (1) silence (knockout) the B2M gene in iPSCs, destabilizing canonical HLAs; (2) insert (knockin) the HLA-E gene to prevent NK cell-mediated cell lysis; or (3) silence the CIITA gene to reduce T cell activation and immune rejection. However, destabilizing the HLA system makes cells susceptible to NK cell attack. Therefore, we silenced CIITA and B2M genes and inserted LLT1A and UL40 genes - inhibitory NK cell membrane proteins found in tumors and human cytomegalovirus, respectively - into human iPSCs using CRISPR/Cas9 and CRISPR-associated transposons. The aim of this project is to characterize HLA-I/B2M knockout and LLT1/UL40 knockin hiPSCs at the gene, transcript, and protein levels, and to determine their ability to evade human T cells, NK cells, and macrophages.