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

Abstract

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 ( hESC s) and human induced pluripotent stem cells ( iPSC s). 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 ( PFF s) to seed the formation for Lewy‐like pathology as measured by phosphorylation of serine‐129 of α‐synuclein ( pS 129‐αSyn). Wild‐type neurons were fully susceptible to the formation of protein aggregates positive for pS 129‐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.

Initiation and propagation of α-synuclein aggregation in the nervous system

“…These findings have been replicated by many studies and in other species, such as rats and primates [20][21][22][23][24][25][26]. Importantly, fibril exposure of human neuronotypic cells derived from differentiated induced pluripotent cells (iPSCs) induces the formation of α-synuclein inclusions and toxicity [27][28][29][30]. These studies demonstrate the potential of human cell models as a platform for screening compounds that potentially mitigate the formation of α-synuclein aggregation and progression of PD.…”

Are PARKIN patients ideal candidates for dopaminergic cell replacement therapies?

“…Using CRISPR/Cas9, we have deleted one or two alleles of the SNCA gene from human embryonic stem cells (hESCs). Upon differentiating, the modified hESCs into mDA neurons and challenging them with α‐synuclein PFFs, we demonstrated that SNCA +/− or SNCA −/− neurons exhibit partial or full resistance to the formation of Lewy‐like pathology (Chen et al., ). This strategy works equally well with induced pluripotent stem cells (iPSCs).…”

Genetically engineered stem cell‐derived neurons can be rendered resistant to alpha‐synuclein aggregate pathology

“…We discuss the implications of a recent study by Chen and collaborators which demonstrated that dopamine neurons derived from human pluripotent stem cells which had been genetically engineered to delete the alpha‐synuclein gene are resistant to the experimental induction of Lewy pathology (Chen et al., ). Neural transplants in Parkinson's disease patients can develop Lewy pathology over a decade after the graft surgery, and this pathology might compromise the long‐term survival and function of the grafted neurons.…”

“…In this issue, Chen and coworkers took another approach (Chen et al., ). They used CRISPR/Cas9 technology to generate human embryonic stem cells (hESC) and iPSCs with deletion of one or both alleles of the alpha‐synuclein gene.…”

“…Taken together, the study by Chen and collaborators is an elegant and state‐of‐the‐art demonstration of CRISPR/Cas9‐mediated deletion of the alpha‐synuclein gene in human stem cells (Chen et al., ). These cells will definitely be welcome laboratory tools for those who are studying mechanisms and consequences of propagation of alpha‐synuclein aggregates, and can be viewed as a “control” paradigm because the cells seem incapable to generating alpha‐synuclein aggregates.…”