567. CRISPR-Cas9 Mediated Gene Editing in a Monogenic Form of Alzheimer's Disease
Authors: Bence Gyorgy, Martin Ingelsson, Camilla Loov, Shuko Takeda, Lars Lannfelt, Bradley T. Hyman, Xandra O. Breakefield
Journal: Molecular Therapy (2016)

Background Individuals heterozygous for the Swedish mutation of the amyloid precursor protein (APPswe) display an increased β-secretase cleavage leading to higher Aβ levels - both in brain and peripheral tissues, such as fibroblasts and plasma. The mutation is a double base change adjacent to each other and has a dominant effect. We hypothesize that the CRISPR system would selectively disrupt the mutated allele without affecting the wild-type allele. Therefore we set out to use the CRISPR system to disrupt expression of mutant APP to counteract Aβ overproduction in Swedish patient fibroblasts. Methods Human APPswe fibroblasts and non-mutated control fibroblasts from subjects of the same family (n=3 for both mutated and control cell lines) were cultured in DMEM medium with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin. Approximately one million cells were used for transfection with the SpCas9 plasmid together with different guide RNAs (gRNAs) designed to bind either the mutated or non-mutated site with the mutation in the gRNA recognition sequence. Four days after transfection, the GFP positive cells were sorted by FACS, enabling separation of Cas9-transfected from non-transfected cells. DNA was extracted from one fraction of the sorted cells, followed by Sanger sequencing and targeted deep sequencing. Another fraction of the cells was further cultured until they were confluent, after which they were kept for 48 h in FBS free media. ELISAs specific for Aβ40 and Aβ42 were used to measure peptide levels in conditioned media from the various cell populations. Results Sanger sequencing was performed on cells that had been successfully transfected with CRISPR plasmids. On such cells, both the APPswe mutant and wt alleles could be disrupted with gRNAs designed against the mutated and non-mutated sites, respectively. Moreover, these effects appeared to be highly specific as assayed by deep sequencing as we did not find any random mutations on the wt allele with the gRNA targeting the mutated site or vice versa. Most of the genomic changes were deletions, but there were some insertions as well. ELISA measurements indicated robustly decreased levels of both Aβ40 and Aβ42 in conditioned media from both mutated and wt cells treated with the respective gRNAs. Shortening the gRNA led to decreased on-target effectivity. Conclusions With the CRISPR/Cas 9 system we could selectively disrupt both the mutated and wt site in fibroblasts from subjects with and without the APPswe mutation. The adjacent double base change created a unique opportunity to selectively disrupt the mutant allele with one of our gRNAs. Furthermore, we could demonstrate a decreased generation of Aβ40 and Aβ42 in the conditioned media from cells treated with the specific gRNAs. This study provides the first experimental evidence that the CRISPR/Cas9 method could be used to develop a novel treatment strategy against familial forms of Alzheimer's disease caused by dominant mutations.