Basic Biology III
Ongoing degeneration and death of dopaminergic (DA) neurons in the midbrain is the hallmark of Parkinson’s disease (PD), a movement disorder that manifests with tremor, bradykinesia and rigidity. Existing therapies for PD are only palliative and treat the symptoms but do not address the underlying cause. Levodopa, the gold standard pharmacological treatment to restore dopamine, is compromised over time by decreased efficacy and increased side effects. Neurosurgical treatments, such as pallidotomy, thalamotomy and deep brain stimulation are considered after failure of pharmacological treatment. Cell replacement therapy involves grafting cells that produce dopamine into the striatum. The proof of principal of this approach has been demonstrated in human PD patients using human brain tissue. However, stem cells are currently the only potential source of reliable, continuous, homogenous and qualified populations of DA neurons for cell therapy. Likewise, the inherent properties of stem cells make them indispensable for investigations aimed at unraveling disease mechanisms for disease modeling and drug discovery. Although human embryonic stem cells (hESCs) potentially offer an unlimited source of cells for cell therapy, to date variations in the efficiency of dopaminergic cell generation, the heterogeneous cellular composition and supply limitations, prevented this therapeutic approach from moving forward towards clinical application. The downstream genetic control of the DA inductive process has not been fully elucidated. Consequently, we are still unable to control the generation of DA neurons from stem cells in vitro or able to control and predict the number of DA-expressing neurons in vivo after transplantation. Undefined cell populations could lead to unpredictable side effects. Thus, the composition of the graft and the proportion of DA neurons in the cell preparation impact the beneficial outcome. To advance cell replacement therapy and drug discovery for PD patients, applied stem cell biology is critical to determine the composition of the therapeutic cellular product and to control the proportion of dopamine and other lineages present. The only way to achieve this goal is to master the generation of specific neural lineages. In this application we propose to define the molecular determinants controlling the generation of the DA phenotype in the A9 DA neurons, the type of DA neurons that consistently promote functional recovery in animal models of PD. This will enable us to induce not only pure DA neuronal populations but to also control the production and proportion of DA neurons in the human neural stem cell cultures and preparations used for cell transplantation. Knowledge from these studies will unravel the gene network involved in the induction, maintenance and stabilization of the DA phenotype, characteristics that are critical to the success of the therapeutic efficacy of the cells.
Statement of Benefit to California:
We have isolated a human neural stem cell line from embryonic stem cells with midbrain properties that we believe is optimal for developing a potential therapeutic product to treat Parkinson’s disease. However the molecular determinants controlling the generation of the dopaminergic neurons are still not fully understood, which could compromise the success of the product development and the therapeutic strategy. We have proposed three aims to define the core transcriptional regulatory circuit controlling the generation of the A9 dopaminergic neurons, the type of cells that consistently promote functional recovery after transplantation in animals models of PD. This will enable us to induce not only pure dopaminergic neuronal populations but to also control the production and proportion of dopaminergic neurons in the human neural stem cell cultures and preparations for therapeutic use. We believe these experiments will provide the foundation for developing a consistent, predictable, safe and efficacious cellular product for Parkinson’s disease patients. This technology will generate intellectual property that will be made available for California based companies to develop and commercialize. All tools and technologies that we develop will be made available to all Californian based scientists in non-profit and industrial organizations. We expect that the money spent on this project will benefit the California research community and will accelerate the research toward developing an efficacious and safe cellular product for treating Parkinson’s disease.
Project synopsis: The goal of this application is to define molecular mechanisms involved in the generation of A9 dopaminergic (DA) neurons from human embryonic stem cell (hESC)-derived neural stem cells (NSCs). DA neurons are a particularly important cell type, as their transplantation has been shown to promote functional recovery in animal models of Parkinson’s disease (PD), a movement disorder for which curative therapies do not currently exist. Clinical proof of principle for cell replacement therapy in PD has been demonstrated as well. Moreover, hESC-derived NSCs are a potential source of DA neurons for cell therapy. However, the inability to generate pure populations of DA neurons is a barrier to the safe use of hESC-derived products in transplantation therapies. The principal investigator (PI) proposes that in order to overcome this barrier, it is necessary to understand the gene network involved in the induction of the DA neuron phenotype. To accomplish this goal, the PI proposes to investigate the role of essential chromatin remodeling genes (Aim 1) and miRNA species (Aim 2) in DA neuron induction from hESC-derived NSCs and to utilize genome-wide mapping technologies to understand the functional relationship between chromatin remodeling and miRNA genes and DA neuron gene expression (Aim 3). Significance and Innovation: - There is clear value in understanding the process of DA neuron induction, and the production of high quality DA neurons for cell transplantation in PD patients is an unmet clinical need. - Although innovative with respect to technology, the proposed project is likely to offer only an incremental advance rather than having a major impact on the development of DA neuron differentiation protocols. Therefore, despite the importance of this problem, reviewers thought the potential impact to be low. - The reviewers noted that clinical and research evidence does not necessarily indicate that simply generating A9 DA neurons and that controlling the "dose" of these cells is necessary or sufficient for the realization of clinical gain. The contribution of other non-DA neuron cell populations, amongst numerous other factors, may prove equally critical, and the applicant did not sufficiently consider this possibility. Feasibility and Experimental Design: - The applicants provide supportive preliminary data and describe previous work demonstrating their ability to generate A9-like DA neurons from hESCs, albeit in low percentages. This data highlights the fact that the current protocols do not produce pure DA neurons. Thus, analysis of the differentiated DA neurons will involve a heterogeneous mix of cells and there is likely to be considerable noise in the system. This is a major weakness in the experimental plan, as the applicants do not address how they will dissect DA neuron-specific changes from the background noise. - Reviewers disagreed on whether or not the applicant presented a logical experimental plan. Some reviewers considered the experimental design clever and comprehensive while others noted that the overall project rationale is poorly articulated and the lack of clarity and detail in the experimental plan made it difficult to understand how the experiments were to be carried out. - There is limited preliminary data supporting the role for chromatin remodeling factors in DA neuron differentiation, and the data is poorly presented. - Potential problems and alternative plans or methodologies were mostly absent. In particular, the applicants do not adequately discuss what they will do if the proposed factor interactions are not confirmed and there is insufficient discussion of controls within experiments. - For the reasons described above, reviewers were concerned that as written, the overall aims are technically probably achievable but it is doubtful that any significant and meaningful data will ensue from this application. Principal Investigator (PI) and Research Team: - The PI has a wealth of expertise in the proposed research area, although some reviewers noted a modest publication record. - The co-investigators are well known and respected, and the senior research team has the appropriate expertise and access to facilities to carry out the proposed work. However, reviewers noted that the junior research team is still to be named with the exception of one postdoctoral fellow. Responsiveness to the RFA: - The proposal is in scope with the RFA as it is focused on hESC fate determination and the epigenetic and other regulatory aspects underlying the developmental potential of human stem cells.
- Ali Brivanlou