Project: Modeling Parkinson’s disease by iPS technology: generation of human affected dopaminergic neurons and gene disease correction by site-specific integration
Induced pluripotent stem (iPS) cells derived from somatic cells of patients represent an innovative
tool for in vitro modeling of diseases and may provide a source for replacement therapies. In
Parkinson disease (PD), the identification of the pathophysiological mechanisms behind this common
neurodegenerative pathology has been particularly hampered by the lack of genuine in vitro models
and animal models faithful to the human pathology.
Exploiting new generations of viral vectors expressing the reprogramming (Oct4, Sox2 and Klf4)
genes in fibroblasts from skin biopsies of the patients, we aim to establish faithful pluripotent iPS cell
lines from sporadic and various monogenic forms of PD carrying mutations in either SNCA, PINK1,
Parkin, LRRK2, DJ-1 or UCH-L1. In order to create an unprecedented in vitro culture system for PD
affected human dopaminergic (DA) neurons, iPS cells will be differentiated in vitro through the
Shh/Fgf8 stepwise induction protocol. Such patient-specific neurons will provide a system where we
will investigate the mechanisms of genetic PD such as neuronal morphology, Lewy body formation,
mitochondrial dysfunction, cell viability after oxidative and other stressors, interactions between
different disease proteins, as well as electrophysiological deficits. For those experiments requiring a
pure population of DA neurons, Pitx3-GFP iPS cell lines will be generated by means of BAC
transgenesis enabling specific labelling of nigral DA neurons. Thus, pure populations of GFP+
dopaminergic neurons will be utilized for microarray based transcriptome profiling comparing PD
versus control neuronal populations. These results promise to add precious information on both
cellular and molecular pathological mechanisms leading ultimately to understand PD progression.
Further, the establishment of human PD affected neurons will offer a remarkable cell platform for
future systematic drug screenings. Finally, we will exploit an innovative strategy for site-specific
integration of the therapeutic gene based on zinc finger nucleases mediated site-specific integration.
This procedure will complement Parkin mutated iPS cells by targeting the therapeutic gene in a safe
and transcriptionally competent genomic harbour.
Overall, this project has the ambitious task to pioneer new approaches for iPS cell generation and
combining them to develop safe gene correction strategies. These new procedures represent a big
advantage for a broad functional characterization of iPS-derived dopaminergic neurons affected by
PD. The successful achievements of our goals will disclose new opportunities for disease modelling,
drug-screenings and cell replacements strategies in PD.
Project partner
