Nuclease-dead S. aureus Cas9 downregulates alpha-synuclein and reduces mtDNA damage and oxidative stress levels in patient-derived stem cell model of Parkinson's disease.

Autor:	Sastre D; Stanford University School of Medicine, Department of Pathology, Stanford, CA 94305, U.S.A., Zafar F; Stanford University School of Medicine, Department of Pathology, Stanford, CA 94305, U.S.A., Torres CAM; Stanford University School of Medicine, Department of Pathology, Stanford, CA 94305, U.S.A., Piper D; San Jose State University, Department of Biological Sciences, San Jose, 95192 CA, U.S.A., Kirik D; Department of Experimental Medical Science, Lund University, Lund, Sweden., Sanders LH; Departments of Neurology and Pathology, Duke Center for Neurodegeneration and Neurotherapeutics, Duke University Medical Center, Durham, NC 27710, U.S.A., Qi S; Stanford University, Department of Bioengineering, Stanford, CA 94305, U.S.A., Schüle B; Stanford University School of Medicine, Department of Pathology, Stanford, CA 94305, U.S.A.
Jazyk:	angličtina
Zdroj:	BioRxiv : the preprint server for biology [bioRxiv] 2023 Jan 24. Date of Electronic Publication: 2023 Jan 24.
DOI:	10.1101/2023.01.24.525105
Abstrakt:	Parkinson's disease (PD) is one of the most common neurodegenerative diseases, but no disease modifying therapies have been successful in clinical translation presenting a major unmet medical need. A promising target is alpha-synuclein or its aggregated form, which accumulates in the brain of PD patients as Lewy bodies. While it is not entirely clear which alpha-synuclein protein species is disease relevant, mere overexpression of alpha-synuclein in hereditary forms leads to neurodegeneration. To specifically address gene regulation of alpha-synuclein, we developed a CRISPR interference (CRISPRi) system based on the nuclease dead S. aureus Cas9 (SadCas9) fused with the transcriptional repressor domain Krueppel-associated box to controllably repress alpha-synuclein expression at the transcriptional level. We screened single guide (sg)RNAs across the SNCA promoter and identified several sgRNAs that mediate downregulation of alpha-synuclein at varying levels. CRISPRi downregulation of alpha-synuclein in iPSC-derived neuronal cultures from a patient with an SNCA genomic triplication showed functional recovery by reduction of oxidative stress and mitochondrial DNA damage. Our results are proof-of-concept in vitro for precision medicine by targeting the SNCA gene promoter. The SNCA CRISPRi approach presents a new model to understand safe levels of alpha-synuclein downregulation and a novel therapeutic strategy for PD and related alpha-synucleinopathies. Competing Interests: Additional information The authors declare no conflict of interest.
Databáze:	MEDLINE
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