Base editing strategy for insertion of the A673T mutation in the APP gene to prevent the development of AD in vitro

• Published in: Molecular therapy. Nucleic acids Vol. 24; pp. 253 - 263
• Main Authors: Guyon, Antoine, Rousseau, Joël, Bégin, Francis-Gabriel, Bertin, Tom, Lamothe, Gabriel, Tremblay, Jacques P.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 04.06.2021; American Society of Gene & Cell Therapy; Elsevier
• Subjects: A673T; Alzheimer's disease; Base editing; CRISPR/Cas9; FAD; Islandic mutation; Original; FAD; CRISPR/Cas9; Alzheimer's disease; Base editing; A673T; Islandic mutation
• ISSN: 2162-2531; 2162-2531
• DOI: 10.1016/j.omtn.2021.02.032

The amyloid precursor protein (APP) is a transmembrane protein mostly found in neurons. Cleavage of this protein by β-secretase can lead to the formation of amyloid-β (Aβ) peptide plaque, which leads to Alzheimer’s disease. Genomic analysis of an Icelandic population that did not show symptoms of Alzheimer’s at an advanced age led to the discovery of the A673T mutation. This mutation can reduce β-secretase cleavage by 40%. We hypothesized that the insertion of this mutation in patients’ neurons could be an effective and sustainable method of slowing down or even stopping the progression of Alzheimer’s disease. We modified the APP gene in HEK293T cells and in SH-SY5Y neuroblastoma using a Cas9 nickase (Cas9n)-deaminase enzyme to convert the alanine codon to a threonine. Several Cas9n-deaminase variants were tested to compare their efficiency of conversion. The results were characterized and quantified by deep sequencing. We successfully introduced the A673T mutation in 53% of HEK293T cells alongside a new mutation (E674K), which seemed to further reduce Aβ peptide accumulation. Our approach aimed to provide a new strategy for the treatment of Alzheimer’s and in so doing, demonstrate the capacity of base editing techniques for treating genetic diseases. [Display omitted] The A673T mutation is associated with neuroprotection against Aβ peptide secretion, leading to plaque buildup in Alzheimer’s disease. Base editing allows this mutation to be introduced into the human cell genome and has shown a decrease in Aβ peptides in vitro.