Projects

Effect of CRISPR-Cas9-induced mutations in the Aire gene (APS1 syndrome) on protein conformation, mTEC cell transcriptome and their interaction with thymocytes.

Geraldo A. Passos (Coordinator)

Eduardo A. Donadi (Principal Investigator)

Thematic Project (2019 – 2024) funded by São Paulo Research Foundation (Fapesp, Grant No. 2017/10780-4)

ABSTRACT

The main objective of the current lab project is to evaluate the effect of pathogenic mutations of the Autoimmune regulator (Aire) gene, the same mutations found in patients with the APS1 autoimmune syndrome (Aire SAND and PHD1 domains), on the following aspects: 1) medullary thymic epithelial cell (mTECs) transcriptome, 2 (mTEC-thymocyte adhesion, thymocyte –> mTEC migration and antigen presentation by mTECs).

Our research group works with the expression of Autoimmune regulator (Aire) gene and “promiscuous gene expression” (PGE) in murine medullary thymic epithelial cells (mTECs). We evidenced that changes in Aire expression are associated with changes in PGE, post-transcriptional control PTAs and the emergence autoimmune type 1 diabetes in NOD mice.

Additionally, we assigned two “new” functions to the Aire gene: 1) regulation of miRNA expression in mTECs and 2) control of mTEC-thymocyte adhesion. In addition, we observed some interesting properties of mTECs; 1) when these cells are cultured together with thymocytes (co-culture as a model system for mTEC-thymocyte adhesion), there is an increase of Aire expression as well as CD80, MHC-II and Fezf2 and 2) mutation induction within the Aire exon 3 through-CRISPR-Cas9 system disturbs the appearing of the Aire protein in the nucleus of mTECs and reduces the adhesion of these cells with thymocytes.

These findings have opened important perspectives to better understand the biology of mTECs and explore points that still are elusive. Therefore, we have formulated the following hypotheses: 1) mTEC-thymocyte adhesion changes the transcriptome (mRNAs and miRNAs) of mTECs and 2) point mutations in the nucleotide sequence of the Aire gene (natural variants), the same mutations found in patients with APS1 (Aire PHD domain on exon 7). Such changes might have a functional consequence in mTEC cells, e.g. changes in the transcriptome and / or mTEC-thymocyte adhesion and / or thymocyte migration towards mTECs.

In this project, we make use of RNA-Seq and microarrays and as well as the mTEC-thymocyte adhesion model (murine model) and  thymocyte –> mTEC transwell migration to test these hypotheses. The technological innovation of the project is the use of CRISPR-Cas9 system for induction of “human” Aire mutations in murine mTEC cells.

We will isolate mTEC Aire mutant clones to evaluate the expression of this gene, the modulation of the transcriptome (mRNAs and miRNAs) using RNA-Seq and / or microarrays. The in vivo experiments will be carried out with a Rosa26-Cas9 knockin transgenic mouse that constitutivelly expresses Cas9 enzyme in all organs and tissues to induce Aire patogenic mutations directly in the thymus.

The scientific innovation is related to the transcriptional response of mTECs when these cells adhere to thymocytes and how Aire mutations could be associated, even indirectly, with the mTEC-thymocyte adhesion, which is the key point for negative selection, the central immunological tolerance and preventing autoimmune diseases.