CRISPR/Cas9 Mediated Disruption of the Swedish APP Allele as a Therapeutic Approach for Early-Onset Alzheimer’s Disease

Abstract

The APPswe (Swedish) mutation in the amyloid precursor protein (APP) gene causes dominantly inherited Alzheimer’s disease (AD) as a result of increased β-secretase cleavage of the amyloid-β (Aβ) precursor protein. This leads to abnormally high Aβ levels, not only in brain but also in peripheral tissues of mutation carriers. Here, we selectively disrupted the human mutant APPSW allele using CRISPR. By applying CRISPR/Cas9 from Streptococcus pyogenes, we generated allele-specific deletions of either APPSW or APPWT. As measured by ELISA, conditioned media of targeted patient-derived fibroblasts displayed an approximate 60% reduction in secreted Aβ. Next, coding sequences for the APPSW-specific guide RNA (gRNA) and Cas9 were packaged into separate adeno-associated viral (AAV) vectors. Site-specific indel formation was achieved both in primary neurons isolated from APPSW transgenic mouse embryos (Tg2576) and after co-injection of these vectors into hippocampus of adult mice. Taken together, we here present proof-of-concept data that CRISPR/Cas9 can selectively disrupt the APPSW allele both ex vivo and in vivo—and thereby decrease pathogenic Aβ. Hence, this system may have the potential to be developed as a tool for gene therapy against AD caused by APPswe and other point mutations associated with increased Aβ.

Applying gene‐editing technology to elucidate the functional consequence of genetic and epigenetic variation in Alzheimer’s disease

“…The resulting increase in secreted Aβ40 and Aβ42 from this mutation leads to severe amyloid pathology (63). Using patient derived fibroblasts carrying the Swedish mutation, Gyorgy et al recently disrupted the cleavage site and observed a 60% decrease in Aβ production (22). Furthermore, the mutation was also targeted in both cultured primary cortical neurons and in vivo using Tg2576 adult mice that carry the APP SW mutation via adeno-associated virus mediated delivery of the Cas9 and sgRNA constructs into the hippocampus.…”

Clinical applications of CRISPR‐based genome editing and diagnostics

“…Extensive work in mice and human cell lines has demonstrated that CRISPR can be used to correct mutations causing Duchenne muscular dystrophy, which recently advanced to restoring dystrophin protein expression in a canine model of the disease through systemic and intramuscular delivery of adeno-associated virus-encoded Cas9. 22 CRISPR-based treatments are also being developed for several brain diseases, 26,[97][98][99] eye diseases such as congenital blindness, and diseases of the liver. 100 The ideal genome-editing therapeutic would allow a single dose of a transiently active nuclease that can be administered systemically but with tissue-selective uptake, editing only the specific cell type in need of correction.…”

Adeno‐associated virus‐based Alzheimer's disease mouse models and potential new therapeutic avenues

“…They found specific disruption (i.e., indel formation) in 1.3% of APPswe alleles in all ( n = 5) injected mice. For comparison, the rate of indel formation was much higher in fibroblasts from human APPswe carriers, which carry only two copies in their genome (György et al, ). This proof‐of‐concept study highlighted the possibility of gene targeting as a potential avenue for treating familial AD and the potential role of AAVs herein.…”