Engineering synucleinopathy-resistant human dopaminergic neurons by CRISPR-mediated deletion of the SNCA gene


An emerging treatment for Parkinson's disease (PD) is cell replacement therapy. Authentic midbrain dopaminergic (mDA) neuronal precursors can be differentiated from human embryonic stem cells (hESCs) and human induced pluripotent stem cells (iPSCs). These laboratory-generated mDA cells have been demonstrated to mature into functional dopaminergic neurons upon transplantation into preclinical models of PD. However, clinical trials with human fetal mesenchephalic cells have shown that cell replacement grafts in PD are susceptible to Lewy body formation suggesting host-to-graft transfer of α-synuclein pathology. Here, we have used CRISPR/Cas9n technology to delete the endogenous SNCA gene, encoding for α-synuclein, in a clinical-grade hESC line to generate SNCA+/- and SNCA-/- cell lines. These hESC lines were first differentiated into mDA neurons, and then challenged with recombinant α-synuclein preformed fibrils (PFFs) to seed the formation for Lewy-like pathology as measured by phosphorylation of serine-129 of α-synuclein (pS129-αSyn). Wild-type neurons were fully susceptible to the formation of protein aggregates positive for pS129-αSyn, while SNCA+/- and SNCA-/- neurons exhibited significant resistance to the formation of this pathological mark. This work demonstrates that reducing or completely removing SNCA alleles by CRISPR/Cas9n-mediated gene editing confers a measure of resistance to Lewy pathology. © 2018 The Authors. European Journal of Neuroscience published by Federation of European Neuroscience Societies and John Wiley & Sons Ltd.

Authors Chen Y, Dolt KS, Kriek M, Baker T, Downey P, Drummond NJ, Canham MA, Natalwala A, Rosser S, Kunath T
Journal The European journal of neuroscience
Publication Date 2019 Feb;49(4):510-524
PubMed 30472757
PubMed Central PMC6492083
DOI 10.1111/ejn.14286

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