Periodic Reporting for period 4 - TREAT-PD (Patient-specific treatment for Parkinson's disease using reprogrammed skin cells)
Periodo di rendicontazione: 2023-05-01 al 2023-10-31
In Parkinson's disease (PD) dopamine neurons are lost and cell therapy, where the lost neurons are replaced by new, healthy cells presents a possibility to tackle the disease This project focus on clinical development of a new reprogramming approach to generate patient specific cells for cell therapy. In the first period we have succeeded in making functional dopamine neurons from adult skin cells. We have started to evaluate if patient derived neurons acquire a disease-related pathology after neural conversion in vitro and after transplantation in vivo. Once completed, this dataset will be used to determine which PD patients would be suitable for autologous grafting, and which would benefit most from cells obtained from matched donors. In subsequent periods, the quality of the reprogrammed dopamine neurons will be stringently assessed in pre-clinical animal models of Parkinson’s Disease. We will compare their properties with that of human fetal dopamine neurons and stem cell derived dopamine neurons in order to ascertain that they are nearly identical to the dopamine neurons you would find in a healthy brain.
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.
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.
At this point, the project has already advanced beyond state of the art in several areas:
1) We have developed a new method that allow for single cell and single nucleus transcriptomic analysis of human cells that have been grafted to the rat brain at a timepoint where they have reached functional maturation. The new method have already resulted in identification of a previously unknown cell type in the grafts and allowed for studies transcriptional diversity of human DA neurons
2) We have developed a reprogramming system that allow for efficient generation of functional DA neurons from patient derived fibroblasts. This new reprogramming method uniquely revealed baseline disease-relevant pathology in the reprogrammed neurons. Importantly, our cellular model also showed that iNs from different patients are not impaired to the same degree and shows that that the degree of impairment relates, at least to some extent, to the age of the donor, the age of onset of disease, and Tau haplotype. This type of age and gene related pathology has not been modelled before, and within this study could not be replicated using iPSCs from the same individuals. This puts forward direct conversion into iDANs to be used for differential diagnostics, drug screening and disease modeling of late onset neurogenerative diseases and also allows for selection of patients most suitable for autologous vs HLA matched grafts.
3) We have established a humanized transplantation model of PD that better recapitulates the main disease features (Hoban et al. PNAS 2020). This reports on a new, fully humanized transplantation model of PD that better recapitulates the main disease features, obtained by co-injection of preformed human a-synuclein (a-syn) fibrils and AAV virus expressing human wild-type a-syn unilaterally into the rat substantia nigra (SN). This is the first pre-clinical model to assess spread of pathology from host to graft, which has previously been reported in transplanted patients but not been able to recapitulate in animal models. This will be of fundamental importance for pre-clinical assessment when developing patient specific treatments for PD.
4) We have completed analysis of DA neurons derived from genetic and sporadic PD patients and shown that these cells are more prone to develop pathology in vitro, in organoids and after transplantation
5) We have shown that cells made disease resistant by knocking out SNCA (the gene coding for asyn which is the main component of lewy bodies) is a viable strategy for autologous grafting.
1) We have developed a new method that allow for single cell and single nucleus transcriptomic analysis of human cells that have been grafted to the rat brain at a timepoint where they have reached functional maturation. The new method have already resulted in identification of a previously unknown cell type in the grafts and allowed for studies transcriptional diversity of human DA neurons
2) We have developed a reprogramming system that allow for efficient generation of functional DA neurons from patient derived fibroblasts. This new reprogramming method uniquely revealed baseline disease-relevant pathology in the reprogrammed neurons. Importantly, our cellular model also showed that iNs from different patients are not impaired to the same degree and shows that that the degree of impairment relates, at least to some extent, to the age of the donor, the age of onset of disease, and Tau haplotype. This type of age and gene related pathology has not been modelled before, and within this study could not be replicated using iPSCs from the same individuals. This puts forward direct conversion into iDANs to be used for differential diagnostics, drug screening and disease modeling of late onset neurogenerative diseases and also allows for selection of patients most suitable for autologous vs HLA matched grafts.
3) We have established a humanized transplantation model of PD that better recapitulates the main disease features (Hoban et al. PNAS 2020). This reports on a new, fully humanized transplantation model of PD that better recapitulates the main disease features, obtained by co-injection of preformed human a-synuclein (a-syn) fibrils and AAV virus expressing human wild-type a-syn unilaterally into the rat substantia nigra (SN). This is the first pre-clinical model to assess spread of pathology from host to graft, which has previously been reported in transplanted patients but not been able to recapitulate in animal models. This will be of fundamental importance for pre-clinical assessment when developing patient specific treatments for PD.
4) We have completed analysis of DA neurons derived from genetic and sporadic PD patients and shown that these cells are more prone to develop pathology in vitro, in organoids and after transplantation
5) We have shown that cells made disease resistant by knocking out SNCA (the gene coding for asyn which is the main component of lewy bodies) is a viable strategy for autologous grafting.