Development of a novel human embryonic stem cell (hESC) model of familial amyotrophic lateral sclerosis (ALS)
ALS is a devastating disorder that lacks effective treatments. Although animal models of the disease have provided some insight into disease mechanism, therapeutic agents identified in animal studies have failed to be useful in ALS patients. Therefore, new human disease models are necessary that take advantage of recently developed high-throughout research methods to advance basic research and the discovery of new therapies for ALS. This proposal aims to develop and investigate a new and unique model of ALS. Human embryonic stem cell (hESC) lines expressing sod-1 mutants linked to familial ALS will be generated for the first time. The following specific aims will be accomplished: 1. A viral vector, will be used to express human wild type or mutated G93A, A4V or I113T sod-1 DNA and a fluorescent protein as a reporter for sod1 expression. An expression enhancer will ensure that SOD-1 and the fluorescent protein are only expressed in cells differentiating to neurons, thereby allowing an easy identification of SOD-1 expressing nerve cells and motor neurons. Cells are differentiated to motor neuron lineage using established protocols. 2. An initial characterization of the new sod-1 transgenic cell lines will include studies of SOD-1 expression, cell death and cell structure to analyze the effect of the mutants on the differentiating and mature motor neurons. 3. A comprehensive high-throughput screen of thousands of small pharmacological compounds will be performed with the goal to identify new therapies for ALS. This hESC model is extremely versatile because it allows the generation and maturation/aging and subsequent examination of cell types, such as motor neurons and the cells surrounding motor neurons (glia) that are affected by ALS. This proposal addresses the major cell type involved in ALS pathology, motor neurons, and outlines a pharmacological screen. Beyond the scope of this proposal, this hESC model holds the promise of learning much more about cellular pathways involved in ALS. The cell lines can be studied at any stage during cell differentiation. The proposed hESC lines will provide an unprecedented tool not only for examining early cellular changes leading to ALS-related cell loss. They will also present a new tool of studying crucial interactions of motor neuron with the cells surrounding them, which are a key to understanding the complex disease mechanisms of ALS. The overall long-term goal of this project is to provide these cell lines to the ALS research community to advance basic and therapy-oriented research for the benefit of ALS patients. The PI has experience with ALS and frontotemporal dementia research, but is new to hESC research. Two collaborators have joined the team, Dr. Harley Kornblum and Dr. Steven Goldman, to provide expert advise and support the successful completion of the project. A postdoctoral fellow, who has proven his skills in the required cell culture techniques, completes the capable team.
Statement of Benefit to California:
This proposal aims to advance ALS research and help to bring about desperately needed new therapies for ALS patients in California. To accomplish this goal, a new human embryonic stem cell (hESC) based cell line will be generated and investigated. This first hESC model of ALS will be useful for innumerous studies that explore the mechanisms underlying ALS and search for new therapies. This work will originate in the state of California, thus strongly supporting the notion that the state of California is a driving force in hESC research in the United States. The derived hESC lines will be shared and used by other laboratories in the country, their California origin will always place a spotlight on the research support that the state of California gives to this cutting-edge technology and on the research accomplishments that can be achieved in this state. As the proposed ALS cell lines will be a versatile and useful tool in ALS research, it is expected that subsequently funding mechanisms from federal and private sources will be obtained for future projects involving the lines. In that case, the state of California would have jump-started an innovative and groundbreaking use of human stem cell technology modeling ALS. In addition, the advancement of biotechnology through future expansion of the proposed project will likely lead to increased hiring of laboratory personnel and the potential of developing therapeutic products with subsequent tax revenues. This specific project will significantly enhance basic ALS research in California by supporting cutting-edge stem cell technology in an institution with a dedicated ALS clinic. California has only very few ALS basic research groups, and no group that combines basic research with clinical ALS work. The combination of ALS basic research and clinical work would be unique and have the potential to give California a leading edge in combining basic and clinical ALS research that is currently held by a few East Coast institutions.
This proposal is designed to establish a model of familial ALS in vitro in motor neurons derived from human embryonic stem cells (hESC’s) that carry mutations in the SOD-I gene. The applicant will firstly transfect hESC’s with a lentiviral vector containing the mutated SOD-I gene and express GFP as a reporter. Motor neurons will be developed from these cells. She will then examine the expression of the mutant gene and its effect on motor neuron survival, and morphology. Finally, she will use high-throughput screening to attempt to identify small molecules that could be therapeutic candidates in ALS. SIGNIFICANCE AND INNOVATION: If successful, this will provide a novel human system in which to explore the biochemical/molecular mechanisms of motor neuron death in ALS. Despite the fact that familial ALS is rare compared to the sporadic disease, there are likely to be shared common pathways of cell death. The question remains as to how different human motor neurons will be from transgenic mouse cells bearing SOD-I mutation nonetheless it would seem worthwhile to establish the mutation in the target cell in ALS, i.e. in human cells. STRENGTHS: • This proposal targets an important neurological disease, ALS. • The proposal is straightforward • The rationale for preparing hESC-derived motor neuron cells that express mutant SOD1s is a good one. These cells will be potentially useful ones for the neurological community • The PI, an Assistant Professor in Department of Neurology since 2005, has expertise in the field of ALS • The collaboration of Drs. Kornblum and Goldman are valuable WEAKNESSES: • The assumption that underlies Aim III is that motor neurons will die following expression of mutant SOD1. However, embryonic motor neurons taken from the CNS of mutant SOD1 transgenic mice have little if any phenotype. It is only with vast overexpression of mutant SOD1 that motor neurons (and other cells) die, perhaps for reasons that are different from those that are present in the mutant SOD1 transgenic mice. If there is no phenotype following mutant SOD1 expression in hESC-derived motor neurons, then there is no rationale for the drug screen. If there is a phenotype following a non-physiological overexpression of mutant SOD1 expression in hESC-derived motor neurons, the positive drug hits may not really be helpful in identifying drugs that will be effective in the SOD1-mediated disease or in sporadic ALS. • There is abundant data emphasizing non-cell autonomous degeneration in familial ALS. Will this phenomenon interfere with the cells showing a phenotype? It would have been appropriate for the PI to have discussed this issue. • The PI brings up cell number as a possible complicating and limiting factor. It would be valuable for this issue to have more discussion . • The PI has had a modest productivity; the most recent first authored scientific paper that was published was in 1996 DISCUSSION: The major concern in the discussion was whether the PI would be able to adequately oversee and perform this work. The PIs record of productivity over the last ten years is weak and it appears that it will be necessary to find others with the necessary skill to perform the work.