Recommended if funds allow
Statement of Benefit to California:
SYNOPSIS: The PI’s overall aim is to develop a model of Alzheimer’s disease (AD) that recapitulates the complex array of human proteins involved in the disease to clarify the pathogenesis as well as provide a cell culture-based system for high-throughput screening for therapies. A rationale for the use of the hES cells is that the rodent Aβ is different biochemically and biophysically from human Aβ and does not form fibrils. In aim 1, the PI will stably transduce two hES cell lines with a lentiviral vector that contains human wild type or mutant APP ; these cells will then be differentiated towards a neuronal lineage and screened with an Aβ ELISA. In aim 2, the PI will utilize AMAXA nucleofection (or, alternatively, lentivrial shRNA delivery)of the hESCs to perform a high-throughput siRNA screen that targets 7,309 genes considered to be potential therapeutic targets, specifically attempting to identify targets that modify the generation or degradation of Aβ as determined by Aβ ELISA or affect Aβ aggregation as determined by thioflavin S fluorescence. INNOVATION AND SIGNIFICANCE: This proposal is aimed at finding a cell culture model system for AD and to use this system for screening in order to identify new therapeutic targets in the disease. Considering the huge and growing problem of AD in the world, the proposal addresses a very significant world health problem. The need of having a reliable in vitro drug screening system (and an in vivo animal model) for screening for candidates drugs for AD is indisputable. At present, there are a number of cell culture models for AD, but they fall short for varied reasons, especially because they generally involve cell lines. The use of hESC-derived neuronal cells that express wild type or mutant APP will be of value since the cells will have a differentiated neuronal phenotype. In addition, the fact that these cells are human makes the cell culture model system a more relevant one than the use of cells from other species. There were differing opinions on the innovativeness of the proposal. One reviewer found the idea of using an ES cell that expresses hAPP as a model system for AD and as a means of screening for therapeutic targets to be an original approach. In addition, the use of siRNAs to screen for the targets takes advantage of very contemporary cutting-edge technology. Another reviewer believed that expressing APP under its native promotor control does not equate to establishing a clone suitable for AD drug screening and found the innovative aspect of the proposed work is low. STRENGTHS: The proposal investigates a significant health problem. The PI is a highly productive investigator who is highly recognized as a leader in the AD field. The collaboration of Dr. Loring, Director, Stem Cell Resource Co-Director, Human Embryonic Stem Cell Center, is a valuable one. The proposal is well-written and well organized. The work contained in the proposal is ambitious but feasible. The PI addresses some potential problems that may arise and some alternative approaches. The lab is well qualified to analyze the amyloid precursor protein (APP) proteolytic products and has expertise in genetic modification of cells. It is likely that transfectants will be obtained. The use of the siRNA approach provides an unbiased screen for therapeutic targets. The work will draw upon knowledge gained from other model systems but will provide novel and useful information about the pathogenesis of AD. WEAKNESSES: The proposal is a risky one; for example, the ELISA screening assay may not be sensitive enough to detect the small levels of Aβ in the siRNA screen described in specific aim 2. The PI notes a "personal communication" from Dr. Loring noting that APP is well expressed in hES cell lines and upregulated upon neuronal differentiation," but does not provide any supporting data. The use of a differentiated hippocampal neuron or another neuronal cell type targeted in AD would be preferable to the more generic differentiated neuron - so that the results of this study may not be similar to those one would find in an AD transgenic mouse One possible concern is that mutant APP will be toxic or have a specific phenotype preventing differentiation. In fact, some investigators have hypothesized that there is a failure in neurogenesis in Alzheimer's disease causing much of the symptomatology. However, a failure of the cells to differentiate may not be without interest. Other concerns expressed included the lack of experience working with hESC and with high through-put screening especially involving siRNA transfection. DISCUSSION: There was discussion on the cell target for siRNA transfection (hESC-derived neurons); on the sensitivity of the ELISA - think may run into trouble with heterogenity; and on the technical capability of the lab for this type of screening. PROGRAMMATIC REVIEW: The Working Group voted to recommend this application for special consideration for funding should additional funds become available based on: 1) it was the only Alzheimer's disease-focused proposal; and 2)the applicant is a very accomplished researcher in this area.