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

Yixi Chen,Marco Kriek,Susan Rosser,Maurice A Canham,Terry Baker,Ammar Natalwala,Tilo Kunath,Patrick Downey,Nicola J Drummond,Karamjit Singh Dolt

doi:10.1111/ejn.14286

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 (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.

Highlights

Parkinson’s disease (PD) is a common neurological disorder that is caused by the loss or dysfunction of specific neuronal cell types
These clinical observations highlight the need for cell therapies that are resistant to the formation of Lewy bodies, which are predominantly composed of the protein α-s ynuclein (Spillantini et al, 1997)
Image quantification of the pS129-αSyn area demonstrated a significant reduction of the Lewy-like structures in SNCAnull neurons (Figure 7c), which is in agreement with the observation in human embryonic stem cells (hESCs)-d erived neurons

Summary

| INTRODUCTION

Parkinson’s disease (PD) is a common neurological disorder that is caused by the loss or dysfunction of specific neuronal cell types. The burden of Lewy bodies in the graft correlated with a decrease in the symptomatic benefit to the patient (Chu & Kordower, 2010) These clinical observations highlight the need for cell therapies that are resistant to the formation of Lewy bodies, which are predominantly composed of the protein α-s ynuclein (Spillantini et al, 1997). The differentiation of human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs) into midbrain dopaminergic (mDA) neurons is well understood and described (Kirkeby et al, 2017; Kriks et al, 2011), and the route to clinical trials for hESC/iPSC-d erived mDA cell therapy for Parkinson’s has been mapped out (Barker, Parmar, Studer, & Takahashi, 2017) These grafts will express wild-type levels of α-synuclein protein making them susceptible to synucleinopathy with a similar kinetics to fetal mesencephalic grafts. This work sets out a strategy to produce disease-resistant mDA grafts for Parkinson’s that will last the life time of the individual, and will be important for patients with an aggressive synucleinopathy

| MATERIALS AND METHODS

| RESULTS

| DISCUSSION

Findings

DATA ACCESSIBILITY