Generation of SNCA Cell Models Using Zinc Finger Nuclease (ZFN) Technology for Efficient High-Throughput Drug Screening

Abstract

Parkinson’s disease (PD) is a progressive neurodegenerative disorder caused by loss of dopaminergic neurons of the substantia nigra. The hallmark of PD is the appearance of neuronal protein aggregations known as Lewy bodies and Lewy neurites, of which α-synuclein forms a major component. Familial PD is rare and is associated with missense mutations of the SNCA gene or increases in gene copy number resulting in SNCA overexpression. This suggests that lowering SNCA expression could be therapeutic for PD. Supporting this hypothesis, SNCA reduction was neuroprotective in cell line and rodent PD models. We developed novel cell lines expressing SNCA fused to the reporter genes luciferase (luc) or GFP with the objective to enable high-throughput compound screening (HTS) for small molecules that can lower SNCA expression. Because SNCA expression is likely regulated by far-upstream elements (including the NACP-REP1 located at 8852 bp upstream of the transcription site), we employed zinc finger nuclease (ZFN) genome editing to insert reporter genes in-frame downstream of the SNCA gene in order to retain native SNCA expression control. This ensured full retention of known and unknown up- and downstream genetic elements controlling SNCA expression. Treatment of cells with the histone deacetylase inhibitor valproic acid (VPA) resulted in significantly increased SNCA-luc and SNCA-GFP expression supporting the use of our cell lines for identifying small molecules altering complex modes of expression control. Cells expressing SNCA-luc treated with a luciferase inhibitor or SNCA siRNA resulted in Z’-scores ≥ 0.75, suggesting the suitability of these cell lines for use in HTS. This study presents a novel use of genome editing for the creation of cell lines expressing α-synuclein fusion constructs entirely under native expression control. These cell lines are well suited for HTS for compounds that lower SNCA expression directly or by acting at long-range sites to the SNCA promoter and 5’-UTR.

Applications of genome editing technology in the targeted therapy of human diseases: mechanisms, advances and prospects

“…After correction, the mtDNA damage disappeared in differentiated neural progenitor and neural cells derived from iPSCs. 134,135 Additionally, Soldner et al 136 combined genome-wide epigenetic information with CRISPR/Cas9 genome editing to generate a genetically precisely controlled experimental system in human iPSCs. This system has identified PD-associated risk variants in noncoding distal enhancer elements that regulate SNCA expression; it has also confirmed that the transcriptional disorder of SNCA is related to sequence-dependent binding of the brain-specific transcription factors EMX2 and NKX6-1.…”

Activators of Alpha Synuclein Expression Identified by Reporter Cell Line-based High Throughput Drug Screen

“…Treatment with valproic acid, a known histone deacetylase inhibitor (HDAC), induces hyperacetylation of global histone H3 at the SNCA promotor and leads to an increase of Snca in rat cerebral granule cells, cortex and cerebellum [ 13 ]. Additionally, VPA was shown to increase SNCA mRNA and α-syn protein levels in SH-SY5Y cells [ 14 ]. Vice versa, reduced H3K27 acetylation marks across the SNCA promotor resulted in decreased SNCA mRNA levels [ 3 ].…”

Establishment of IL-7 Expression Reporter Human Cell Lines, and Their Feasibility for High-Throughput Screening of IL-7-Upregulating Chemicals

“…Additionally, we show that the reporter system can be used to screen chemicals inducing IL-7 gene upregulation in an HTS format. Although our HTS design for identifying small molecules that upregulate endogenous target gene transcription is not commonly used [ 38 , 39 ], our screening scheme and pilot screening performance was found to be good and robust; we obtained a good z′-factor (HTS assay feasibility index) and z-factors (HTS quality index) [ 26 ] and identified the EL, an IL-7 upregulator, through small-scale screening. In addition, we found that EL upregulated IL-7 gene transcription strongly in A549 lung cells and weakly in several other cancer cell lines.…”