Early Translational I
Parkinson’s disease (PD) is a devastating movement disorder caused by the death of dopaminergic neurons, a specific subtype of neurons in the brain. These neurons secrete dopamine (a signaling molecule) and among higher brain functions are a critical component of the motor circuit that ensures movements are smooth and coordinated. All current treatments attempt to overcome the loss of these neurons by either replacing the lost dopamine, or modulating other parts of the circuit to balance this loss or attempting to halt or delay the loss of dopaminergic neurons. Cell replacement therapy (e. g. transplantation of dopaminergic neurons into the brain to replace lost cells and restore function) as proposed in this application attempts to use cells to release small amounts of dopamine to a specific brain area to provide appropriate activation of the affected motor circuitry. It is firmly established from experimental evidence in animal studies and human trials that dopaminergic cell replacement is a realistic strategy. However, some clinical studies have reported only variable success. Most transplantation studies into Parkinson patients have used cells from tissue chunks of human fetal brains pooled from multiple aborted fetuses. We therefore conclude from these results that a therapeutic dopaminergic cell type is present in these grafts but their precise identity is not known (perhaps pliable dopamine progenitor cells engraft better than mature neurons). Likely, these studies grafted variable proportions of dopamine neurons without control over the presence of other, potentially counteracting, neuronal subtypes. To date there are no ways to purify and specifically test dopamine neurons or their precursor cells. This is the key stumble block that hinders the development of an investigational new drug (IND) for cell replacement in PD. We will therefore first develop methods to isolate and functionally test dopamine neurons and their progenitors and thereby identify and refine the development candidate for successful cell therapy. The isolation of a drug from aborted human embryos is complicated by enormous logistic and technical complications, ethical considerations, and potential immunerejection of the graft. We will use induced pluripotent stem (iPS) cells derived from the patients’ own skin to derive defined and genetically-matched dopamine neuronal populations in a scalable manner. In addition, from the same iPS cells we will generate transplantable neural cells to serve as small, regulated pumps of growth factors that have been shown to inhibit dopamine neurons from degenerating but lack of efficient delivery has hindered clinical application. In this proposal, we have assembled a strong California team of stem cell biologists, developmental neurobiologists, clinicians, and a for-profit company whose combined efforts are realistic to develop novel, preclinical candidates of individualized cell-based therapies for PD.
Statement of Benefit to California:
We have assembled a team of scientists and clinicians that aim to develop a cell transplantation therapy for a currently non-curable disease, Parkinson’s disease, using patients’ skin-derived pluripotent stem cells. We believe that this proposal includes the basic elements that are required for the translation of basic research to clinical research. We have proposed to obtain pluripotent stem cells from skin in a safe manner which is prerequisite of Food and Drug Administration (FDA)-approval for clinical use of this important stem cell type. This will provide California’s academic and commercial community with the potential to generate these pluripotent stem cells from patients suffering from a variety of different diseases in a way that is compatible for clinical use to treat a number of diseases including Diabetes, spinal chord injury, and genetic skin and muscle diseases. Further, we plan to develop two therapeutic cell populations which will be tested in preclinical studies for efficacy and safety and refined to become candidate drugs to be tested in further clinical settings. Although transplantation will not cure many of the more general symptoms associated with Parkinson’s disease, specific features of the movement disorder will be responsive to the treatment. As currently 1-2% of the population older than 65 years is diagnosed with this devastating neurodegenerative disease, any progress towards relieving the major motor symptoms of Parkinson patients will be of immense benefit to the State of California and its citizens. In addition, all the tools and reagents that we develop will be made widely available to Californian researchers and we have selected a California-based company for potential commercialization. We hope that California-based physicians will be at the forefront of developing this promising avenue of research. We expect that the money expended on this research will benefit the Californian research community and the tools and reagents we develop will help accelerate the research of our colleagues in both California and worldwide.
The overall goal of this proposal is to derive two therapeutic cell populations from autologous pluripotent stem cells for the treatment of Parkinson’s disease (PD). In a first specific aim, the applicant will generate “safe” induced pluripotent stem cells (iPSCs) from PD patients using a system that promotes single-site integration of DNA plasmids into defined genomic sites and allows for subsequent excision of these sequences. These iPSCs will be characterized for pluripotency and compared by gene expression to other pluripotent cell lines. In the second aim, the applicant will derive human dopaminergic (DA) neurons from iPSCs, test their engraftment and functionality by transplantation into rat and mouse models of PD and develop methods for producing clinical-grade DA neurons for human transplantation. In the final aim, the applicant proposes to generate astrocytes engineered for inducible secretion of glial derived neurotrophic factor (GDNF). After deriving these cells from iPSCs, their therapeutic potential will be tested by transplantation into rat and mouse models of PD. Reviewers agreed that the proposed research could have significant impact. PD is a major unmet clinical challenge and current pharmacological therapies treat only symptoms and lose clinical efficacy over time. The field is in great need of disease-modifying therapies. Cell-based therapies for PD have tremendous potential, and long-term sustained clinical benefit has been seen in some patients transplanted with human fetal mesencephalic tissue. But given the profound shortage of human fetal tissue of the appropriate age, this treatment will never benefit more than a small number of patients, and DA neurons derived from stem cells could provide an unlimited source of cells appropriate for central nervous system (CNS) transplantation. There is also evidence that delivery of growth factors that are neurotrophic and neuroprotective for DA neurons may have therapeutic efficacy for some PD patients, so astrocytes derived from iPSCs and genetically engineered to express GDNF may also provide a new therapeutic avenue for PD. One reviewer questioned whether the proposal is truly translational, describing it as a series of basic research projects scattered along the translational trajectory, and felt that the proposed studies had little potential to rapidly advance novel cell therapies to the clinic. Reviewers found the proposal’s research plan to be of limited feasibility, overly ambitious and lacking essential detail. They noted that most of the proposed work is highly descriptive and devoid of experimental methodology, description of controls, time lines, and verifiable outcomes. The use of the proposed system to generate more genetically defined iPSCs is a novel approach, but much of this work, as well as that surrounding the generation of human DA neurons for PD therapy, is poorly developed. There were no preliminary data presented on the differentiation protocols for either DA neurons or astrocytes, and the efficacy of the proposed protocols has not been demonstrated by other groups. Reviewers felt that much of the work described in the proposal is highly speculative and problematic. For example, in Aim 2, the applicant proposes to insert genes for several different transcription factors (which may be important in DA lineage specification) into mouse embryonic stem cells through homologous recombination and into human iPSCs via bacterial artificial chromosomes; these gene transfer methods have a very low efficiency of transfer and are notoriously difficult. The applicant provides no methodology for how she/he intends to accomplish this difficult task. Furthermore, it is unclear whether the applicant intends to deliver all of these genes to the same sets of cells or establish individual cell lines. In the subsequent analysis, the applicant proposes to perform “genome-wide” expression profiling of these cell populations in order to generate a candidate list of cell-surface proteins, but details were not provided about how expression profiling will be performed or how the list of proteins will be prioritized and validated. Reviewers found the application abundant in similar experiments for which insufficient detail was provided. Reviewers praised the applicant’s background and publication record but were concerned about his/her lack of experience running projects of this size. Reviewers appreciated the strength of the assembled research team but noted that some of the collaborating investigators’ roles in the project were poorly defined. For example, the roles of two investigators were described as to “oversee experiments related to guidance molecule expression”, but there was very little in the research plan relating to such experiments. Reviewers felt that the large number of investigators committing to only 1-5% effort was a significant weakness. Additionally, reviewers found the budget to be inappropriately high and a number of consultant costs largely unjustified. Overall, although reviewers appreciated the potential impact of this proposal, they found the research plan to be overly ambitious in scope and lacking rigor and important experimental details.