5 research outputs found
Engineering synucleinopathyâresistant human dopaminergic neurons by CRISPRâmediated 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 laboratoryâgenerated 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 hostâtoâgraft transfer of αâsynuclein pathology. Here, we have used CRISPR/Cas9n technology to delete the endogenous SNCA gene, encoding for αâsynuclein, in a clinicalâgrade hESC line to generate SNCA+/â and SNCAâ/â cell lines. These hESC lines were first differentiated into mDA neurons, and then challenged with recombinant αâsynuclein preformed fibrils (PFFs) to seed the formation for Lewyâlike pathology as measured by phosphorylation of serineâ129 of αâsynuclein (pS129âαSyn). Wildâtype neurons were fully susceptible to the formation of protein aggregates positive for pS129âαSyn, while SNCA+/â and SNCAâ/â neurons exhibited significant resistance to the formation of this pathological mark. This work demonstrates that reducing or completely removing SNCA alleles by CRISPR/Cas9nâmediated gene editing confers a measure of resistance to Lewy pathology