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Molecular basis of disease-associated differentiation dysfunction in single cells

Funding Type: 
Basic Biology V
Grant Number: 
Funds requested: 
$1 476 048
Funding Recommendations: 
Not recommended
Grant approved: 
Public Abstract: 
Despite years of research, little is known about the molecular mechanisms that contribute to neurodevelopmental disorders such as autism, and currently there are few therapeutic options. To understand such diseases and develop successful therapies, we need for accurate disease models. Culture of disease-relevant cell types offers the opportunity to investigate aspects of the disease that are impossible to study in people or mouse models. Differentiation of human pluripotent stem cells has been shown to recapitulate many aspects of human development, including the molecular cues required for brain development. We have generated iPSCs (induced pluripotent stem cells) from several patients clinically diagnosed with Fragile X Syndrome, a leading genetic cause of autism. Our Fragile X-iPSC lines exhibit aberrant neurogenesis, a phenotype consistent with other neurodevelopmental disorders. We propose to correct the disease-causing mutations in our patient stem cell lines, thereby generating genetically matched control cell lines. Using state of the art global genome and gene expression profiling techniques, we will build a molecular model of the events that occur during neural differentiation of Fragile X-iPSCs. Results of these studies will provide a detailed understanding of the molecular events underlying neurodevelopmental disorders, identify dysfunctional genes/pathways in Fragile X Syndrome, and identify molecular targets with therapeutic potential.
Statement of Benefit to California: 
The incidence of autism and autism spectrum disorders cases diagnosed in California and worldwide has increased dramatically in recent years. The molecular mechanisms that contribute to neurodevelopmental disorders such as autism remain elusive, and currently there are no therapeutic treatments for autism. We propose to use patient-specific induced pluripotent stem cells and state of the art genetic analysis techniques to build a molecular map of Fragile X Syndrome, the most common form of inherited autism. Results of our study will benefit the citizens of California by increasing our knowledge about the mechanisms underlying neurodevelopmental disorders and identifying molecular targets with therapeutic potential.
Review Summary: 
This Fundamental Mechanisms Track application investigates the molecular basis of the neuronal defects associated with Fragile X syndrome (FXS). FXS is a genetic syndrome and can cause mental retardation and autism, especially in young boys. In Aim 1, undifferentiated and differentiated FXS patient-derived induced pluripotent stem cells (iPSCs) will be analyzed using gene expression and epigenetic profiles. Aim 2 will examine the molecular differentiation of individual cells by using a novel single cell transcriptome profiling. Aim 3 will assess the effects of removing a repeat sequence from a critical gene implicated in FXS. Significance and Innovation - The significance and innovation of the proposed studies were considered moderate - The proposed single cell sequencing for analyzing gene expression was considered innovative Feasibility and Experimental Design - The application does not address the cause of neuronal impairment, which could arise due to factors such as cell fate, neuroblast proliferation, or delayed differentiation. - Reviewers criticized that appropriate controls have not been used. For instance, FXS iPSCs have not been compared with iPSCs from aged-matched healthy donors for their ability to generate neurons. Preliminary data only show a comparison of iPSCs with established human embryonic stem cells. - The technology to remove the trinucleotide repeats by gene targeting, though well-established in mature cell lines, may not work well in iPSCs. - Reviewers stated that details regarding data interpretation and application were lacking and suggested that the PI increase the emphasis on data analysis. Principal Investigator (PI) and Research Team - The application lacks valuable expertise in neuroscience that will be very helpful to streamline the defects in the FXS neurons. - The applicant has a strong track record and has significant collaborations with primary experts in the fields of pluripotent stem cells, reprogramming, genomics and bioinformatics analyses. - State-of-the-art equipment and facilities are available and of great support for this project. Responsiveness to the RFA - The proposal is responsive to the RFA as it investigates the molecular basis of a relevant neuroinfantile disorder through iPSC modeling and genome-wide technologies.
  • Sean Palecek
  • Todd C. McDevitt

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