Genome Editing of α-Synuclein in iPSCs from a Donor with Multiple System Atrophy

Multiple system atrophy (MSA) is a neurodegenerative disorder of primarily glial origin. Clinically it is distinct from Parkinson’s disease with predominantly autonomic failure and motor impairment. Pathologically, the principal cellular targets are oligodendrocytes that show abundant glial cytoplasmic inclusion bodies consisting of -synuclein (SNCA) aggregates and nigrostriatal degeneration. Protein misfolding and aggregation of alpha-synuclein is a common feature across synucleinopathies and an attractive target for drug development. In order to study the contribution of -synuclein to the disease phenotype of MSA, we generated induced pluripotent stem cells (iPSCs) from a donor with MSA and then created isogenic lines with SNCA deletions using the GeneArt ® Precision TAL-nuclease fusion technology. We designed two sets of TALEs to create deletions in exon 2 of the SNCA gene in patient derived iPSCs via non-homologous end-joining (NHEJ). After functional verification of the SNCA TALENs carried out in HEK and U-2 OS cells, and confirmed with the Surveyor assay and Sanger sequencing, iPSCs from the MSA donor were edited with SNCA TALENs. Colonies that showed positive cuts in the Surveyor assay were picked and expanded. These colonies were further characterized by next gensequencing on the Ion Torrent PGM instrument to identify clonal populations with SNCA deletional mutations. The edited clones with SNCA heterozygous deletion mutation are karyotypically normal and express pluripotency markers. These clones are in the process of re-editing to generate homozygous deletions, after which they will be further differentiated into appropriate cell types for phenotypic studies. In conclusion, TAL technology can be applied to generate isogenic disease iPSCs for studying the contribution of SNCA in the disease phenotype of MSA.