The impact of ADA2 downregulation on T-cell functioning of CRISPR/Cas9 edited PBMCs
Abstract
Adenosine Deaminase 2 Deficiency (DADA2) is a rare autosomal genetic syndrome characterized by auto-inflammatory symptoms, where the development of T lymphocytes and B lymphocytes is malfunctioning. The deficiency is caused by the loss of function mutations in the CECR1 gene, which encodes the enzyme adenosine deaminase 2. Adenosine Deaminase 2 (ADA2) is involved in the metabolism of adenosine and has a role in regulating the immune response, and in addition it has been linked to the regulation of T-cell activation and differentiation. Recent studies suggest that downregulation of ADA2 can lead to altered T-cell activation and proliferation. The pathogenesis of the disease is still not completely understood.
The aim of this study was to study the effect of knock out of ADA2 and knock in of ADA2 R169Q mutation on the activity and expression of the ADA2 gene. and study the impact of ADA2 downregulation on T-cells functioning. The editing on T-cells was done to understand more of the mechanism of DADA2. Electroporation was used as delivery method for CRISPR/Cas9 in PBMCs (Peripheral Blood Mononuclear Cells) to knock out the ADA2 gene and knock in a mutation of ADA2 (R169Q), a mutation linked to the development of ADA2 deficiency. Further the cells were analyzed to evaluate ADA2 enzyme activity and gene expression, and the effect of ADA2 knockout and R169Q mutation on T-cells were studied using flow cytometry in T-cell activation and proliferation downstream assays.
A decrease in ADA2 activity and gene expression were observed for the ADA2 KO and KI of R169Q samples compared to the non-edited samples. However, for the study of T-cell functioning, some problems occurred for long cultivation of the edited T-cells, which was further discussed and compared with cells undergoing a shorter cultivation period.
Thus, future experiments are needed with a wider set of samples from multiple donors with enhanced editing efficiency. It is crucial to optimize the experimental procedures for utilizing edited cells to gain deeper insights into the effects of ADA2 downregulation on T-cell functionality. Taking these factors into consideration will contribute to a more comprehensive understanding of the impact of ADA2 downregulation on the functioning of T-cells.