Optimization of <i>in vitro</i> gene delivery to neurons containing an APP gene with a mutation responsible for familial Alzheimer’s disease for the development of a base editing therapy

Abstract Background The Amyloïd Precursor Protein (APP) is preferentially cut by the α‐secretase enzyme in healthy individuals. However, an abnormal cleavage by β‐secretase leads to the accumulation of β‐amyloid peptides. Numerous APP genetic mutations cause Familial Alzheimer Disease (FAD). However, a rare APP gene variant (A673T) found in Icelanders drastically reduces cleavage by the β‐secretase. We hypothesized that the insertion of this A673T mutation in a patient’s genome using the CRISPR/Cas9 Base‐Editing Technology (Komor et al. Nature 2016) could be an effective method to slow down the progression of familial and sporadic forms of Alzheimer’s disease. The objective of this study is to eventually compare the efficiency by which a lentiviral or a dual‐AAV‐based vector could deliver a base‐editing complex (SpCas9VQR‐Target‐AID) into neurons in vitro. Method Neurons were obtained using two methods. The first method involved differentiating fibroblasts from a FAD patient or a mouse model with a London (V717I) mutation into neurons (Shrigley et al. JoVE 2018) (Figure 1), while the second method involved isolating hippocampal neurons from prenatal NL/F/G mice (Seibenhener et al. JoVE 2012). The dual AAV complex was designed to deliver two parts of the base editor gene separated by inteins (Villiger et al. Nat Med 2018). Hippocampal or induced neurons were transduced with lentivirus or AAV1. Cas9 detection was performed by immunofluorescence and Western Blot. The β‐amyloid peptides in supernatant were characterized and quantified by Meso Scale Discovery’s Aβ kit. Result We have been able to detect the Cas9 protein in neurons by western blot and immunofluorescence (Figure 2). In subsequent experiments, we will determine which delivery method will reduce more the β‐amyloid peptide. Conclusion Our approach aims to confirm the protective effect of the A673T mutation against the development of familial and sporadic forms of Alzheimer’s disease as well as develop an efficient delivery method for the base editing complex which will lead to an in vivo application. Long‐term perspective: We aim to develop a completely new therapeutic approach for AD and FAD using base editing or the new PRIME editing technique (Anzalone et al. Nature 2019). This may eventually lead to a Phase I clinical trial.

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