Browsing by Subject "Base editing"

Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is an inherited autosomal dominant disease that leads to cognitive impairment, vascular dementia and ischemic strokes. In CADASIL, vascular smooth muscle cells (VSMCs) degrade gradually and are replaced by connective tissue in the small and mid-sized arteries in the brain. Extracellular granular osmiophilic material (GOM) that surround the VSMCs are a unique feature in CADASIL. The causal gene behind CADASIL is Notch3, which encodes a transmembrane protein with a signaling function. There are over 200 cysteine-altering mutations that cause CADASIL in Notch3. The potential pathology causing mechanism is still unclear, but most likely the mechanism is linked to the aggregation of GOM deposits that are potentially toxic to VSMCs. This thesis project aimed to correct CADASIL causing c.475C>T mutation in Notch3 in different CADASIL cell lines with different CRISPR base editor systems. Another aim was to create induced pluripotent stem cell (iPSC) lines from a CADASIL patient-derived skin biopsy sample to be used in the creation of an in vitro disease model for CADASIL. RNA-based ABEmax base editor system was used to correct immortalized- and primary- CADASIL cell lines. DNA-based ABEmax base editor system was used as a positive control. Simultaneous pluripotent reprogramming and pathogenic CADASIL mutation correction were done in the same transfection during this project. The editing efficiencies were evaluated by Sanger sequencing the genomic target region before and after the transfection. The editing efficiencies were good in general compared to literature. They ranged from 27 % to 73 % target base editing efficiency depending on the editing system-, guide-RNAs - and electroporation parameters used. Confirmed proximal off-target effects were not detected, and distal off-target effects were not evaluated.