Tools and Technologies II
Two main applications of stem cells in regenerative medicine involve cell replacement therapies and building human “disease-in-a dish” models to reveal underlying cellular and molecular events and to enable drug screenings. One of the major challenges of using “disease-in-a-dish” models is to reveal physiologically-relevant phenotypes and to use the phenotype for drug screening. Over the past several years, our laboratory has been working actively towards using human stem cell–based “disease-in-a-dish” models to study human neurological disorders. We have found that at least for neurological diseases, two universal assays can be applied. One is electrophysiological analysis for sensitive detection of abnormalities in synaptic transmission , as vast majority of neurological disorders involve abnormalities in neural transmission, and the other is alterations in transcriptome. Since it is increasingly acknowledged that the transcriptome of a cell to a large extent, represents the biological/physiological state of that cell, it is becoming meaningful to utilize the transcriptome as a general readout that marks the epigenetic state of a cell, which may reflect its pathological state, identity, differentiation and physiological state. Once the whole genome-wide transcriptome is reduced to a core/signature program of a handful of genes, there is a highly sensitive and quantitative medium-throughput screening platform, which can be used for core-transcriptome reversal screening that can also be considered as an universal phenotypic reversal screening. This application is aimed at testing the validity of this approach and if successful, it will greatly accelerate the drug discovery process using the human disease-in-a-dish models for neurological disorders, paving the way from disease genes all the way to therapeutic development.
Statement of Benefit to California:
Rett Syndrome (RTT) is a progressive neurodevelopmental disorder caused by primarily loss-of-function mutations in the X-linked MeCP2 gene. It mainly affects females with an incidence of about 1 in 10,000 births. After up to 18 months of apparently normal development, children with RTT develop severe neurological symptoms including motor defects, mental retardation, autistic traits, seizures and anxiety. RTT is one of the Autism Spectrum Disorders (ASDs) that affects many children in California. In this application, we propose to use our hESC-based Rett syndrome (RTT) model as a proof-of-principle case to define a set of core transcriptome that can be used for drug screenings. Human embryonic stem cells (hESCs) hold great potential for cell replacement therapy where cells are lost due to disease or injury. For the diseases of the central nervous system, hESC-derived neurons could be used for repair. This approach requires careful characterization of hESCs prior to utilizing their therapeutic potentials. Our findings will not only benefit RTT and other ASD patients, but also subsequently enable broad applications of this approach in drug discovery using human pluripotent stem cell-based disease models to benefit the citizens of California in a broader spectrum.
The goal of this proposal is to develop drug targets for Rett syndrome (RTT) by identifying a core set of genes whose expression is altered in a disease-specific manner and to validate this approach as a generalized strategy for drug discovery. RTT syndrome is an autism spectrum developmental disorder caused by a mutation in the MeCP2 gene and associated with abnormal synaptic function in neurons. The applicant has developed an in vitro model for RTT syndrome in which the MeCP2 gene is knocked down in neurons derived from human embryonic stem cells (hESCs); these neurons display an abnormal synaptic phenotype. The applicant has utilized microarray technology to identify a set of genes whose expression is modified in the MeCP2 knock-down neurons. In Aim 1 the applicant will define a core set of relevant genes (core transcriptome). In Aim 2 they proposed to assess the role of these genes as mediators of the aberrant phenotypes in the RTT in vitro model. Finally, in Aim 3, the hESC-derived MeCP2 knock-down neurons will be used to screen a small molecule chemical library to identify compounds capable of reverting aberrant core transcriptome profiles to the normal state. Reviewers found that the concept of a core signature transcriptome responsible for RTT syndrome phenotypes to be novel and interesting and acknowledged that the discovery of a potential therapeutic candidate for this disorder would be of high impact. However they considered the approaches outlined in the proposal to lack innovation and found the rationale obscure. Additionally, reviewers agreed that the lack of feasibility of the experimental plan limited any potential impact of the proposed research. The experimental plan was abbreviated and lacked sufficient experimental detail for the reviewers to discern how the applicant would conduct the proposed studies. For example, quantitative criteria for selecting candidates for the core transcriptome were not provided, criteria for assessing the effect of one gene on another were absent, and a functional index was not described for determining the hierarchy of the core transcriptome candidates in a transcriptional signaling network. Given this lack of information, it was difficult to assess whether or not core transcriptome genes would be identified using the approaches outlined in the proposal. A similar lack of detail in the description of the approaches for small molecule screening rendered assessment of the proposed screening experiments challenging. The applicant proposes to use commercially available chemical libraries, however, no details were provided about these libraries, preventing reviewers from assessing their value. Further, the applicant did not describe how screens would be performed or how hits would be identified. The preliminary data supporting the feasibility of the proposed experiments was weak, and the applicant did not clearly describe how the provided preliminary data supported the proposed experiments. Furthermore, rationale for the experimental design was lacking. The presented timeline was non-specific and unrealistic, and a discussion of milestones was absent from the proposal. The principal investigator (PI) on the proposal is an accomplished, senior neuroscientist with a strong track record and the expertise to perform the experiments described in the proposal. Reviewers considered the overall research team to be inadequate and inexperienced, particularly in drug screening expertise. Overall, although the PI is strong and the proposed research is in an area of great interest, reviewers considered the proposal to be weak and poorly written. The complete lack of experimental detail or scientific rationale for the proposed experiments led reviewers to doubt the feasibility and, therefore, the impact of the proposed research. This application was not recommended for funding.