Dataset Information

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.

SUBMITTER: Gyorgy B

PROVIDER: S-EPMC5992788 | biostudies-literature | 2018 Jun

REPOSITORIES: biostudies-literature

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

György Bence B Lööv Camilla C Zaborowski Mikołaj P MP Takeda Shuko S Kleinstiver Benjamin P BP Commins Caitlin C Kastanenka Ksenia K Mu Dakai D Volak Adrienn A Giedraitis Vilmantas V Lannfelt Lars L Maguire Casey A CA Joung J Keith JK Hyman Bradley T BT Breakefield Xandra O XO Ingelsson Martin M

Molecular therapy. Nucleic acids 20180316

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 APP<sup>SW</sup> allele using CRISPR. By applying CRISPR/Cas9 from Streptococcus pyogenes, we generated allele-specific deleti ...[more]

PMID: 29858078

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Project description:BackgroundA barrier to HIV-1 cure rests in the persistence of proviral DNA in infected CD4+ leukocytes. The high HIV-1 mutation rate leads to viral diversity, immune evasion, and consequent antiretroviral drug resistance. While CRISPR-spCas9 can eliminate latent proviral DNA, its efficacy is limited by HIV strain diversity and precision target cell delivery.MethodsA library of guide RNAs (gRNAs) designed to disrupt five HIV-1 exons (tat1-2/rev1-2/gp41) was constructed. The gRNAs were derived from a conseensus sequence of the transcriptional regulator tat from 4004 HIV-1 strains. Efficacy was affirmed by gRNA cell entry through transfection, electroporation, or by lentivirus or lipid nanoparticle (LNP) delivery. Treated cells were evaluated for viral excision by monitoring HIV-1 DNA, RNA, protein, and progeny virus levels.FindingsVirus was reduced in all transmitted founder strains by 82 and 94% after CRISPR TatDE transfection or lentivirus treatments, respectively. No recorded off-target cleavages were detected. Electroporation of TatDE ribonucleoprotein and delivery of LNP TatDE gRNA and spCas9 mRNA to latently infected cells resulted in up to 100% viral excision. Protection against HIV-1-challenge or induction of virus during latent infection, in primary or transformed CD4+ T cells or monocytes was achieved. We propose that multi-exon gRNA TatDE disruption delivered by LNPs enables translation for animal and human testing.InterpretationThese results provide "proof of concept' for CRISPR gRNA treatments for HIV-1 elimination. The absence of full-length viral DNA by LNP delivery paired with undetectable off-target affirms the importance of payload delivery for effective viral gene editing.FundingThe work was supported by the University of Nebraska Foundation, including donations from the Carol Swarts, M.D. Emerging Neuroscience Research Laboratory, the Margaret R. Larson Professorship, and individual donor support from the Frances and Louie Blumkin Foundation and from Harriet Singer. The research received support from National Institutes of Health grants T32 NS105594, 5R01MH121402, 1R01Al158160, R01 DA054535, PO1 DA028555, R01 NS126089, R01 NS36126, PO1 MH64570, P30 MH062261, and 2R01 NS034239.

Dataset Information

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

Publications

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

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