The objective of this study is to develop a new, optimized technology to obtain a homogenous population of midbrain dopaminergic (mDA) neurons in a culture dish through neuronal differentiation. Dopaminergic neurons of the midbrain are the main source of dopamine in the mammalian central nervous system. Their loss is associated with one of the most prominent human neurological disorders, Parkinson's disease (PD). There is no cure for PD, or good long-term therapeutics without deleterious side effects. Therefore, there is a great need for novel drugs and therapies to halt or reverse the disease.
Recent groundbreaking discoveries allow us to use adult human skin cells, transduce them with specific genes, and generate cells that exhibit virtually all characteristics of embryonic stem cells, termed induced pluripotent stem cells (iPSCs). These cell lines, when derived from PD patient skin cells, can be used as an experimental pre-clinical model to study disease mechanisms unique to PD. These cells will not only serve as an ‘authentic’ model for PD when further differentiated into the specific dopaminergic neurons, but that these cells are actually pathologically affected with PD.
All of the current protocols for directed neuronal differentiation from iPSCs are lengthy and suboptimal in terms of efficiency and reproducibility of defined cell populations. This hinders the ability to establish a robust model in-a-dish for the disease of interest, in our case PD-related neurodegeneration. We will use a new, efficient gene integration technology to induce expression of midbrain specific transcription factors in iPSC lines derived from a patient with PD and a sibling control. Forced expression of these midbrain transcription factors will direct iPSCs to differentiate into DA neurons in cell culture. We aim at achieving higher efficiency and reproducibility in generating a homogenous population of midbrain DA neurons, which will lay the foundation for successfully modeling PD and improving hit rates of future drug screening approaches. Our study could also set a milestone towards the establishment of efficient, stable, and reproducible neuronal differentiation using a technology that has proven to be safe and is therefore suitable for cell replacement therapies in human.
The absence of cellular models of Parkinson’s disease represents a major bottleneck in the scientific field of Parkinson’s disease, which, if solved, would be instantly translated into a wide range of clinical applications, including drug discovery. This is an essential avenue if we want to offer our patients a new therapeutic approach that can give them a near normal life after being diagnosed with this progressively disabling disease.
The proposed research could lead to a robust model in-a-dish for Parkinson’s disease (PD)-related neurodegeneration. This outcome would deliver a variety of benefits to the state of California.
First, there would be a profound personal impact on patients and their families if the current inevitable decline of PD patients could be halted or reversed. This would bring great happiness and satisfaction to the tens of thousands of Californians affected directly or indirectly by PD.
Progress toward a cure for PD is also likely to accelerate the development of treatments for other degenerative disorders. The technology for PD modeling in-a-dish could be applied to other cell types such as cardiomyocytes (for heart diseases) and beta-cells (for diabetes). The impact would likely stimulate medical progress on a variety of conditions in which stem cell based drug screening and therapy could be beneficial.
An effective drug and therapy for PD would also bring economic benefits to the state. Currently, there is a huge burden of costs associated with the care of patients with long-term degenerative disorders like PD, which afflict tens of thousands of patients statewide. If the clinical condition of these patients could be improved, the cost of maintenance would be reduced, saving billions in medical costs. Many of these patients would be more able to contribute to the workforce and pay taxes.
Another benefit is the effect of novel, cutting-edge technologies developed in California on the business economy of the state. Such technologies can have a profound effect on the competitiveness of California through the formation of new manufacturing and health care delivery facilities that would employ California citizens and bring new sources of revenue to the state.
Therefore, this project has the potential to bring health and economic benefits to California that is highly desirable for the state.
The goal of this study is to develop a technology that optimizes current approaches of differentiating induced pluripotent stem (iPS) cells into midbrain dopaminergic (mDA) neurons. This will be accomplished by using an integrase system to facilitate the integration of mDA neuron-specific transcription factors into iPS cells derived from patients with genetic forms of Parkinson’s disease (PD) as well as from control individuals. The goal of Aim 1 is to generate integrase-modified iPS cell lines that carry two or three mDA neuron-specific transcription factors in various combinations. In Aim 2, the applicants will determine if forced expression of these factors improves differentiation of iPS cells to mature mDA neurons when compared to control iPS cells without integrated transcription factors. In their final Aim 3, in order to validate the pathological phenotype of the newly generated PD-specific neurons, they propose to carry out a functional characterization of the mDA neurons derived from the integrase engineered iPS cells compared to neurons derived from control iPS cells.
Loss of mDA neurons is one of the main hallmarks of PD. The absence of cellular models represents a major bottleneck in the scientific field of PD, therefore reviewers agreed that the production of a large number of mDA neurons would potentially have a major impact on PD research. They found the gene delivery approach to be sophisticated and the overall significance of the proposed research to be strong, but noted that the general goal of the project was not innovative since similar research is already being carried out by several groups in California and elsewhere.
Reviewers found the research plan to be well articulated and the rationale for the proposed studies to be sound. Strong preliminary data in the murine system demonstrate facility with the gene delivery system, but reviewers expressed some concern regarding the feasibility of other aspects of the proposal. The proposed gene delivery approach still requires traditional gene targeting and also efficient DNA transfer into cells, both known to be challenging in human pluripotent stem cells, and neither is supported by preliminary data in human cells. Moreover, the proposed experiments related to the generation of a PD disease phenotype in neurons are quite vague and little experimental detail is provided. Reviewers would have appreciated more consideration for characterizing the newly generated mDA neurons since a mixed population of various types of mDA neurons would likely lead to inconclusive results when using the cells as models of PD for drug screening. Finally, a reviewer commented that each of the aims is predicated on the successful achievement of the previous aims, and expressed concern if forced expression of the transcription factors does not yield large numbers of DA neurons, then the rest of the work will be halted. A lack of alternative approaches related to the generation of DA neurons was further noted.
The applicant is a well-established investigator with considerable experience in genomic modifications, and has demonstrated capacity and expertise to lead large research teams. Reviewers also found the research environment to be excellent. Most reviewers agreed that the research team is outstanding and has most of the required expertise to perform the proposed research. However, they suggested the addition of a neuropharmacologist specializing in dopamine receptors and a neuroanatomist to characterize different DA neuron types.
Overall, reviewers agreed that the strengths of the application are its potential impact, the applicant’s expertise, and the preliminary data regarding the gene delivery system. However, enthusiasm was reduced by the lack of detail and alternative approaches in some areas of the experimental plan as well as some concern regarding the feasibility of the project.
- A motion was made to move this application into Tier 1, Recommended for Funding. Reviewers reiterated that the genetic engineering aspects of this proposal were strong, but that the DA neurobiology aspects were less convincing. Reviewers noted that the top tier already contains an application proposing PD iPS cell work, but that it differs significantly in approach. They further made the point that CIRM does not currently have a strong PD portfolio. The motion carried.