The goal of this proposal is to establish a novel research tool to explore the molecular basis of Parkinson’s disease (PD) - a critical step toward the development of new therapy. To date, a small handful of specific genes and associated mutations have been causally linked to the development of PD. However, how these mutations provoke the degeneration of specific neurons in the brain remains poorly understood. Moreover, conducting such genotype-phenotype studies has been hampered by two significant experimental problems. First, we have historically lacked the ability to model the relevant human cell types carrying the appropriate gene mutation. Second, the genetic variation between individuals means that the comparison of a cell from a disease-carrier to a cell derived from a normal subject is confounded by the many thousands of genetic changes that normally differentiate two individuals from one another. Here we propose to combine two powerful techniques – one genetic and one cellular – to overcome these barriers and drive a detailed understanding of the molecular basis of PD. Specifically, we propose to use zinc finger nucleases (ZFNs) in patient-derived induced pluripotent stem cells (iPSC) to accelerate the generation of a panel of genetically identical cell lines differing only in the presence or absence of a single disease-linked gene mutation. iPSCs have the potential to differentiate into many cell types – including dopaminergic neurons that become defective in PD. Merging these two technologies will thus allow us to study activity of either the wild-type or the mutant gene product in cells derived from the same individual, which is critical for elucidating the function of these disease-related genes and mutations. We anticipate that the generation of these isogenic cells will accelerate our understanding of the molecular causes of PD, and that such cellular models could become important tools for developing novel therapies.
Approx. 36,000-60,000 people in the State of California are affected with Parkinson’s disease (PD) – a number that is estimated to double by the year 2030. This debilitating neurodegenerative disease causes a high degree of disability and financial burden for our health care system.
Importantly, recent work has identified specific gene mutations that are directly linked to the development of PD. Here we propose to exploit the plasticity of human induced pluripotent stem cells (iPSC) to establish models of diseased and normal tissues relevant to PD. Specifically, we propose to take advantage of recent developments allowing the derivation of stem cells from PD patients carrying specific mutations. Our goal is to establish advanced stem cell models of the disease by literally “correcting” the mutated form of the gene in patient cells, therefore allowing for direct comparison of the mutant cells with its genetically “repaired” yet otherwise identical counterpart. These stem cells will be differentiated into dopaminergic neurons, the cells that degenerate in the brain of PD patients, permitting us to study the effect of correcting the genetic defect in the disease relevant cell type as well as provide a basis for the establishment of curative stem cells therapies.
This collaborative project provides substantial benefit to the state of California and its citizens by pioneering a new stem cell based approach for understanding the role of disease causing mutations via “gene repair” technology, which could ultimately lead to advanced stem cell therapies for Parkinson’s disease – an unmet medical need without cure or adequate long-term therapy.
This proposal is focused on the generation of induced pluripotent stem cell (iPSC)-derived “disease in a dish” models to explore the molecular basis of Parkinson’s disease (PD). The applicant proposes to use zinc finger nucleases (ZFNs) to genetically induce and correct the most common genetic mutation associated with Parkinsonism. This approach will permit the generation of genetically identical (isogenic) iPSC lines, save the presence of the PD-linked mutation. The applicant identifies that lack of such isogenic control iPSC lines as a translational bottleneck to the development of therapeutics. There are four specific aims: (1) to use ZFNs to correct a specific mutation in PD patient-derived iPSCs; (2) to use ZFNs to knock down this mutation in PD patient-derived iPSCs; (3) to use ZFNs to generate the same PD-linked mutation in iPSCs derived from control individuals; and (4) to differentiate all of the cell lines generated in Aims 1-3 into dopaminergic (DA) neurons and compare their molecular and cellular phenotypes. The overall deliverable in this grant will be the creation of an isogenic panel of iPSCs that carry different allelic forms of the gene of interest at the endogenous locus, thus providing robust genetic tools for repair of disease-specific mutations, drug discovery and disease mechanism research.
The reviewers agreed that this proposal addresses a significant translational bottleneck and could have a major impact if successful. They noted that isogenic cell lines will allow direct comparisons between diseased iPSCs and ideal control cells. Reviewers described the proposal as novel and highly innovative. They appreciated the strong and clear scientific rationale, particularly for Aim 3, in which iPSCs from mutation-free carrying individuals (control) will be targeted with ZFNs to induce the PD-linked mutation of interest. A reviewer described this as an extremely important aim because it will establish whether this mutation can cause the PD phenotype by itself or by acting in concert with other loci.
Reviewers praised the research plan and extremely strong preliminary data. They noted that the four aims are complex and technically challenging but feasible. The applicant has identified a ZFN pair that can cleave DNA an appropriate distance from the targeted mutation, an essential reagent for the proposed studies. This ZFN pair has already been used to induce mutations in patient-derived fibroblasts. While iPSCs will be more difficult to transfect, the applicant has proposed multiple strategies. The reviewers appreciated the thorough discussion of pitfalls and alternative approaches. One reviewer expressed concern that the entire project is dependent on the ability to reproducibly generate large enough quantities of DA neurons to serve as an in vitro model of PD. However, other reviewers noted that the challenge of DA neuron differentiation isn’t restricted to this application and the issue of cell number is less important for in vitro models than it is for in vivo transplantation.
The reviewers described the Principal Investigator (PI) as an experienced and well-regarded leader in the field of PD research. They appreciated the strong collaboration with a private company that has pioneered ZFN technology. One reviewer noted that a biosketch is missing for the staff scientist in charge of DA neuron differentiation, making it difficult to judge the team’s expertise in this area. But, in general, reviewers found the research team well qualified to carry out the proposed experiments.
Overall, the reviewers were enthusiastic about this proposal to generate novel cellular tools to study the molecular basis of PD and perform drug screens. They found the proposal innovative, highly significant and supported by strong preliminary data. Reviewers were impressed by the overall approach and noted that it has the potential to lead to paradigm-shifting discoveries.