Validation of hiPSCs gene editing for the generation of non-immunogenic clones

• Grant number: 23/08319-8
• Support Opportunities: Scholarships in Brazil - Scientific Initiation
• Start date: July 01, 2023
• End date: August 31, 2023
• Field of knowledge: Biological Sciences - Genetics - Human and Medical Genetics
• Principal Investigator: Ernesto da Silveira Goulart Guimarães
• Grantee: Caroline Brandão Chiovatto
• Host Institution: Instituto de Biociências (IB). Universidade de São Paulo (USP). São Paulo , SP, Brazil

Abstract

Induced pluripotent stem cells (iPSCs) are in vitro reprogrammed cell lines derived from fibroblasts or erythroblasts that, like embryonic stem cells (ESCs), have the ability to differentiate into all tissues of an organism. Due to their high proliferative potential and permissiveness to gene editing and clonal selection, iPSCs have great potential for cellular therapies in humans. Immunological rejection is one of the main challenges in the field of cell therapy, where cells of the immune system recognize transplanted cells as foreign and eliminate them. Human leukocyte antigen class I (HLA-I) is primarily responsible for alloimmune rejection, as it presents peptide antigens to CD8+ T cells and initiates the cytotoxic immune response. The beta-2 microglobulin (B2M) protein stabilizes class I HLA molecules on the cell surface. To avoid CD8+ T cell-mediated immunological rejection, it is possible to: (1) silence (knockout) the B2M gene in iPSCs, destabilizing canonical HLAs; (2) insert (knockin) the HLA-E gene, avoiding NK cell-mediated cellular lysis; or (3) silence the CIITA gene, reducing T cell activation and immunological rejection. However, destabilizing the HLA system may compromise the immune system's ability to recognize and eliminate tumor cells and virus-infected cells. In our laboratory, we have silenced the HLA-I and B2M genes and inserted the LLT1A and UL40 genes - derived from human cytomegalovirus - into human iPSCs using CRISPR/Cas9 and CRISPR-associated transposons. The objective of this project, therefore, is to characterize the HLA-I/B2M knockout and LLT1A/UL40 knockin hiPSCs at the gene, transcript, and protein levels.