Nuclease-dead S.aureusCas9 downregulates alpha-synuclein and reduces mtDNA damage and oxidative stress levels in patient-derived stem cell model of Parkinson’s disease

Abstract

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.

Robust nuclease-dead S. aureus dCas9-mediated alpha-synuclein knockdown in substantia nigra in a humanized mouse model of Parkinson’s disease

“…SadCas9 is one of the smaller Cas9 proteins which are developed for in vivo gene therapy applications. We also showed that lowering alpha-synuclein in human iPSC-derived neuronal cultures from a patient with an SNCA triplication is reducing mitochondrial DNA damage and oxidative stress levels suggesting a reduction in disease burden 38 .…”

“…Based on our previous in vitro screen for nuclease-dead S. aureus Cas9 (sadCas9) sgRNAs in the human SNCA promoter 38 , we tested five sgRNAs around the transcriptional start site 2 (TSS2) of the human SNCA gene (TSS2-sg1 to TSS2-sg5) with homology to non-human primates ( Figure 1A,B ). HEK293T cells were triple transfected with the doxycycline-inducible SadCa9-RFP, sgRNA-BFP, and reverse tetracycline-controlled transactivator construct ( Figure 1C ) and yielded a transfection efficiency of 50-60% after FACS sorting ( Figure 1E ).…”

“…This study is built on two previous publications that showed evidence for proof of concept that the SNCA promoter can be successfully targeted by CRISPR/Cas9 interference 30,49 . Heman-Ackah et al found one sgRNA in the region of the transcriptional start site 2 (TSS2) of the SNCA promoter that showed a 50% SNCA downregulation in HEK293 cultures and human iPSC-derived neurons.…”

“…In that study, the S. pyogenes version of CRISPR/Cas9 was used which is 4.2 kb in length. In our previous in vitro study 49 and in this study, we used S. aureus dCas9 guided to the promoter region of SNCA gene which also robustly mediated reduction of mRNA and protein levels in vitro. SadCas9 is one of the smaller Cas9 proteins which are developed for in vivo gene therapy applications.…”

A blood-based marker of mitochondrial DNA damage in Parkinson’s disease

“…Many promising compounds or treatments targeting mitochondrial impairment in PD are in the pipeline (66,67). Yet, to date, a functional mitochondrial blood marker that could be used to establish a PD mitochondrial subtype that reflects this underlying pathophysiology has not been developed, thereby allowing a precision medicine-based approach to PD clinical trials, which may yield a higher chance of success (37,68). Our data provide evidence to support inclusion of mtDNA damage as a blood-based candidate marker for PD in future clinical trials.…”