Correction of a Signal Transducer and Activator of Transcription 1 (STAT1) gain-of-function mutation in T cells by applying the CRISPR-Cas9 technique

Abstract

Signal Transducer and Activator of Transcription 1 (STAT1) gain-of-function is a primary immunodeficiency disease caused by a mutation in the STAT1 gene. STAT1 gain-of-function leads to a hyperphosphorylation of STAT1. The disease is characterized by immune dysregulation, increased risk of bacterial, viral, and fungal infections, chronic mucocutaneous candidiasis, autoimmunity, and inflammation. There are no satisfactory treatment options for STAT1 gain-of-function.

Gene therapy by precise genome editing has shown promising results in treating monogenic diseases. The clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9) technique offers a simple and effective way of correcting disease-causing mutations. Mutation correction occurs as a result of homology-directed repair (HDR), where Cas9 and guide RNA initiate a precise double-stranded DNA break, and a repair template containing the correction of the mutation is perfectly integrated into the target DNA.

The aim of this thesis was to correct the STAT1 gain-of-function mutation in a patient's T cells in vitro to an extent where it improved the cell functionality by using the CRISPR-Cas9 technique. T cells from the patient were transfected by electroporation with Cas9, guide RNA, and repair template to correct the STAT1 gain-of-function mutation. Three guide RNAs were screened for the highest level of mutation correction. The best-performing guide RNA was used to screen for the most efficient mutation correction when combined with seven repair templates. The level of mutation correction by HDR was measured by droplet digital PCR.

Flow cytometry analysis of phosphorylated STAT1 was performed on the optimized combination of guide RNA 2 and repair template 4 to investigate whether the level of mutation correction, measured by a reduced amount of phosphorylated STAT1, improved the functionality of the patient’s T cells. The amount of phosphorylated STAT1 was reduced in the corrected patient T cells compared to that of uncorrected patient T cells.

The findings of this thesis suggest that correcting the STAT1 gain-of-function mutation in a patient’s T cells in vitro by using the CRISPR-Cas9 technique yields a promising improvement in the functionality of the patient’s T cells.