We recently developed a new vector system that for the first time allowed for efficient conversion of fibroblasts from adult and aged individuals into functional induced neurons (Drouin-Ouellet et al. EMBO Mol Med. 2017). During the ERC grant period, we advanced the methodology further, so that subtype specific and functional induced DA neurons (iDANs) can be reprogrammed from adult fibroblasts with high efficiency and purity. Furthermore, we have shown that the reprogramming strategy works on both healthy controls, idiopathic PD patients and genetic PD patients.
During the course of the project however, new studies from my own group as well as other recently published studies have shown the induced neurons maintain the age of the donor. Since this increase the risk of the patient derived cells carrying disease related features, we carefully assessed this in the iDANs from patients and healthy controls. When analyzing the patient-derived neurons, we found that stress-induced chaperone-mediated autophagy and macroautophagy impairments could be detected in the idiopathic PD but not in control iDANs. Indeed, further studies showed that induced DA neurons from idiopathic PD patients accumulated pathological forms of alpha-synuclein (asyn), hallmark of PD, after challenges in vitro (Drouin-Ouellet et al. Stem Cell Reports 2022).
The data reported in this study shows that the cellular system developed is ideal for studying late onset neurodegenerative diseases, but that it carries a risk if used for cell therapy. To mediate this, we work along two major lines in subsequent work 1) gene correction/gene editing and/or 2) use of iPSCs instead of iDANs as a source of patient derived DA neurons for transplantation.
These two approaches have both been carried through to WP2: Evaluation of disease-associated pathology WP 3: Pre-clinical evaluation. This has increased the total workload of the project, but has been possible to carry out and deliver results as planned since we established a collaboration with Dr Tilo Kunath at Unversity of Edinburgh that has already cells and material is place for relevant gene editing and gene corrections.
A major achievement in the 3rd period has been assessments of patient derived cells in our new, fully humanized and disease mimicking xenograft model of PD (Hoban et al PNAS 2020; Shrigley et al JPD 2020 and Nilsson et al, submitted). The data from these in vivo studies, as well as the in vitro data reported in (Drouin-Ouellet et al. Stem Cell Reports 2022) show that DA neurons reprogrammed from PD patients are more prone to develop pathology over time and therefore not suitable for use without prior modifications. To make patient derived cells that are resistant to the disease pathology we have explored gene correction for monogenetic lines and as a universal strategy that can be used both for sporadic and genetic lines. We have completed the initial pre-clinical assessment studies of patient derived cells as well as genetically modified disease-resistant patient derived cells and show good graft survival and maturation, unaffected circuitry integration and therapeutic effect (Nilsson et al. submitted). Long term studies in xenograft studies are now ongoing.
