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CRISPR-Cas9 treatment partially restores amyloid-β 42/40 in human fibroblasts with the Alzheimer's disease PSEN1 M146L mutation
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Geriatrics. (Molekylär geriatrik)ORCID iD: 0000-0002-8753-347X
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Geriatrics. (Molekylär geriatrik)
Harvard Med Sch, Massachusetts Gen Hosp, Dept Neurol, Memory Disorders Unit, Charlestown, MA USA..
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Geriatrics. (Molekylär geriatrik)
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2022 (English)In: Molecular Therapy Nucleic Acids, E-ISSN 2162-2531, Vol. 28, p. 450-461Article in journal (Refereed) Published
Abstract [en]

Presenilin 1 (PS1) is a central component of γ-secretase, an enzymatic complex involved in the generation of the amyloid-β (Aβ) peptide that deposits as plaques in the Alzheimer’s disease (AD) brain. The M146L mutation in the PS1 gene (PSEN1) leads to an autosomal dominant form of early-onset AD by promoting a relative increase in the generation of the more aggregation-prone Aβ42. This change is evident not only in the brain but also in peripheral cells of mutation carriers. In this study we used the CRISPR-Cas9 system from Streptococcus pyogenes to selectively disrupt the PSEN1M146L allele in human fibroblasts. A disruption of more than 50% of mutant alleles was observed in all CRISPR-Cas9-treated samples, resulting in reduced extracellular Aβ42/40 ratios. Fluorescence resonance energy transfer-based conformation and western blot analyses indicated that CRISPR-Cas9 treatment also affects the overall PS1 conformation and reduces PS1 levels. Moreover, our guide RNA did not lead to any detectable editing at the highest-ranking candidate off-target sites identified by ONE-seq and CIRCLE-seq. Overall, our data support the effectiveness of CRISPR-Cas9 in selectively targeting the PSEN1M146L allele and counteracting the AD-associated phenotype. We believe that this system could be developed into a therapeutic strategy for patients with this and other dominant mutations leading to early-onset AD.

Place, publisher, year, edition, pages
2022. Vol. 28, p. 450-461
Keywords [en]
MT, RNA/DNA editing, Alzheimer’s disease, presenilin 1, amyloid-β, fibroblasts, CRISPR-Cas9, gene editing, protein conformation, off-target effects
National Category
Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy) Medical Genetics Neurology
Identifiers
URN: urn:nbn:se:uu:diva-475572DOI: 10.1016/j.omtn.2022.03.022ISI: 000795090300005PubMedID: 35505961OAI: oai:DiVA.org:uu-475572DiVA, id: diva2:1664159
Funder
Swedish Research Council, 2018-03075Swedish Research Council, 2021-02793AlzheimerfondenThe Swedish Brain FoundationÅhlén-stiftelsenStiftelsen Gamla TjänarinnorGun och Bertil Stohnes StiftelseNIH (National Institutes of Health), R35 GM118158German Research Foundation (DFG), 417577129NIH (National Institutes of Health), AG044486NIH (National Institutes of Health), AG015379
Note

Title in Web of Science: CRISPR-Cas9 treatment partially restores amyloid-beta 42/40 in human fibroblasts with the Alzheimer's disease PSEN1 M146L mutation

Available from: 2022-06-03 Created: 2022-06-03 Last updated: 2023-11-03Bibliographically approved
In thesis
1. CRISPR/Cas9-based therapies and the role of astrocytes in Alzheimer’s disease and Parkinson’s disease
Open this publication in new window or tab >>CRISPR/Cas9-based therapies and the role of astrocytes in Alzheimer’s disease and Parkinson’s disease
2022 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Alzheimer’s disease (AD) and Parkinson’s disease (PD) are the two most common neurodegenerative disorders. Whereas the AD brain features plaques of amyloid-beta (Aβ) and neurofibrillary tangles of tau, the PD brain is characterized by Lewy bodies and Lewy neurites containing α-synuclein (αSyn). Rare familial disease forms have illustrated a central involvement of these proteins in the respective pathogeneses. Mutations in the genes for the presenilins (PSEN1, PSEN2) result in AD by an increased generation of the more aggregation prone Aβ42 peptide, whereas mutations in the αSyn gene (SNCA) cause PD by affecting aggregation of αSyn.

This thesis has investigated the gene editing tool CRISPR/Cas9 as a potential treatment strategy against AD and PD. When targeting PSEN1 M146L in patient fibroblasts, the increased Aβ42/Aβ40 ratio was partially restored and the treatment typically normalized the mutation-induced conformation of presenilin 1. Moreover, the treatment did not cause any major off-target effects across the genome. For SNCA, both the wild-type form and the A53T mutant were targeted. Lentivirus-mediated delivery of CRISPR/Cas9 to patient fibroblasts and HEK293T cells led to a targeting efficiency of up to 87%. However, treatment of A53T mutant patient fibroblasts only resulted in low and inconsistent targeting efficiencies.

During the course of AD, progressive cellular dysfunction and degeneration cause widespread neuronal death. Apart from neurons, also glial cells are affected by the disease process. Astrocytes, the most abundant glial cell type, play a key role in maintaining brain homeostasis. However, in a neurodegenerative environment, astrocytes enter a reactive and inflammatory state that can potentially harm nearby neurons.

To further investigate the role of astrocytes in AD, we generated a co-culture system of human induced pluripotent stem cell-derived neurons and astrocytes. We observed a differential effect of direct and remote astrocytic control on neuronal viability and functionality. Physical astrocytic contact combined with the presence of Aβ resulted in increased phagocytosis and clearance of dead cells as well as a reduced neuronal activity. However, indirect contact via conditioned media from control astrocytes improved the viability of neurons, whereas addition of Aβ led to hyperactivity. Analyses of long-term astrocytic cultures revealed a persistent reactive state accompanied by a limited Aβ degradation capacity and severe cellular stress.

Overall, this thesis has explored novel gene therapeutic strategies for AD and PD as well as contributed with knowledge regarding the role of astrocytes in AD progression.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2022. p. 70
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 1857
Keywords
Alzheimer's disease, Parkinson's disease, gene editing, CRISPR/Cas9, amyloid-beta, astrocytes, neurons, iPSCs, electrophysiology
National Category
Neurosciences
Identifiers
urn:nbn:se:uu:diva-481628 (URN)978-91-513-1567-6 (ISBN)
Public defence
2022-09-30, Rudbecksalen, Rudbecklaboratoriet, Dag Hammarsköljds väg 20, Uppsala, 09:00 (English)
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Supervisors
Available from: 2022-09-08 Created: 2022-08-13 Last updated: 2022-09-08

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Konstantinidis, EvangelosMolisak, AgnieszkaStreubel-Gallasch, LinnAguilar, XimenaGiedraitis, VilmantasEssand, MagnusErlandsson, AnnaIngelsson, Martin

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Konstantinidis, EvangelosMolisak, AgnieszkaStreubel-Gallasch, LinnKim, Daniel Y.Aryee, Martin J.Aguilar, XimenaGiedraitis, VilmantasEssand, MagnusErlandsson, AnnaIngelsson, Martin
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