Base editing strategy allows insertion of the A673T mutation in APP gene to prevent the development of Alzheimer's disease

Abstract

Amyloid precursor protein (APP), a membrane protein mostly found in neurons, is preferentially cut by the -secretase enzyme, however, abnormal cleavage by {beta}-secretase leads to the formation of {beta}-amyloid peptide plaque in the brains of Alzheimers patients. Genome analysis of an Icelandic population that did not appear to show symptoms of Alzheimers at advanced age led to the discovery of the A673T mutation, reducing {beta}-secretase cleavage by 40%. We hypothesized that the insertion of this mutation in a patients genome could be an effective and sustainable method to slow down or prevent the progression of familial and sporadic forms of Alzheimers disease. We have thus modified the APP gene in HEK293T cells and in SH-SY5Y neuroblastoma using a Cas9n-deaminase enzyme, which changes a cytosine into a thymine, thus converting 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 modified the APP gene in up to 56.7% of the HEK293T cells. Our approach aimed to attest to the efficiency of base editing in the development of treatments against genetic diseases as well as provide a new strategy for the treatment of Alzheimers.