Human-Induced Pluripotent TSC1 and TSC2 Mutant Stem Cell-Derived Neuronal Assays for Mechanistic Studies and Therapeutics Development
Abstract
While tuberous sclerosis complex (TSC) is characterized by benign tumor growth in multiple organs, more than 90% of TSC patients develop neuropsychiatric disorders including epilepsy and autism spectrum disorder (ASD). Studies have suggested defects in early neural progenitor differentiation, cortical connectivity, functions of GABAergic neurons, synapse development and plasticity, neurite outgrowth, and axon specification in TSC. Human samples of TSC have been limited to those obtained during epilepsy surgery. Using TSC-specific induced pluripotent stem cells (iPSCs) and their direct-differentiated neurons offers an unlimited source of human material to recapitulate TSC pathology, to understand TSC disease mechanisms at the cellular level, and to facilitate discovery of novel therapeutics. Eighty-five percent of TSC patients harbor mutations in either TSC1 or TSC2 genes. The clinical disease manifestations produced by TSC1/2 mutations vary from individual to individual, including those within a given family with the same genetic mutation. Hence, isogenic iPSC lines that share the same genetic background except for the TSC1 or TSC2 mutations are needed to isolate the impact of the TSC1/2 mutations. As Co-Principal Investigator (Co-PI) Dr. Wen has done before, we can use gene editing to generate the isogenic TSC1 and TSC2 mutant lines. We have developed efficient protocols to differentiate iPSCs into highly enriched neural progenitor cells, GABAergic and glutamatergic neurons (99%, >80%, 90%, respectively). These cell types are relevant to TSC s neural pathology. We will characterize the cellular defects of the TSC1 and TSC2 mutant neuronal cells and use them as phenotypic screening assays to: (1) screen for new drugs; (2) assess the impact of timing of treatment initiation, dose, and treatment duration of rapamycin, its derivatives, and other new therapies in the pipeline on neuronal differentiation and functions; and (3) study the disease mechanism and identify new drug targets. We can assess the impact of TSC1 and TSC2 mutations on gene expression at single-neuron level, potentially overcome the challenge that neural cells tend to be heterogeneous. Using these data, we will identify gene and pathways that are impacted by the mutations, and potentially identify new biomarkers for diagnostics and disease management as well as new drug targets. Our past work illustrates the success of our proposed approach in mechanistic studies and drug discovery. Co-PI Dr. Wen and JuvoBio scientific co-founder Professors Song and Ming derived iPSCs from schizophrenia patients with a DISC1 mutation. He identified a causal biological pathway and reversed cellular disease phenotypes through pharmaceutical modulation of the pathway. We developed a 384-well HTS assay based on this discovery, and JuvoBio is collaborating with a pharma partner to conduct a screen of 100,000 compounds and with a second pharma partner to test six of its advanced compounds for potential further development for schizophrenia. Even though mTOR inhibitors have been shown to be effective treating lymphangioleiomyomatosis and other TSC symptoms, rapamycin and derivatives are immosuppressants whose long-term use could have negative health impact. In addition, long-term exposure to rapamycin was shown to inhibit both mTOR Complex 1 and mTOR Complex 2, whose functions are multiple and diverse, which could lead to harmful side effects as well. New targets and new drugs will benefit patients. In addition to TSC research and therapy discovery, mechanistic insights gained and reagents and assays generated from this study can also benefit other neurodevelopmental conditions such as ASD and epilepsy.
Document Details
- Document Type
- DoD Grant Award
- Publication Date
- Jan 31, 2017
- Source ID
- W81XWH1610412
Entities
People
- Wei Zhang
Organizations
- United States Army