High throughput multiplexed assay for profiling gene expressions of schizophrenia iPSC-derived neurons

Project Summary High throughput multiplexed assay for profiling gene expressions of schizophrenia iPSC derived neurons This proposal is to develop and validate a secondary high throughput screening HTS assay for new therapeutics for schizophrenia SCZ By assessing primary hit compounds ability to ameliorate transcriptional dysregulation in SCZ neurons and not to dysregulate normal neurons in well format the potential mechanism of actions MOAs of the hits can be quickly assessed in a high throughput and low cost fashion This assay addresses the main shortcoming of phenotypic screens hits of unknown mechanisms We have a validated primary HTS synaptic density assay using forebrain glutamatergic neurons FGNs derived from induced pluripotent stem cells iPSCs bearing a Disrupted in schizophrenia DISC frameshift mutation This proposed secondary screen our primary screen and other confirmatory assays we have developed form a powerful toolset for SCZ drug discovery SCZ a major public health problem affects around of the world s population It manifests with diverse symptoms is challenging to treat has complex genetic risk associations and poorly understood etiology of patients do not respond to medications show partial response and only enter full remission SCZ medications tend to be partially effective for positive symptoms but have limited or no effect for negative symptoms or cognitive impairments which are the most important predictors for long term social functioning Hence new psychotropic therapeutics are need to address significant unmet medical needs We have optimized differentiation protocols that produce highly pure populations of cortical neural progenitors NPCs GABAergic interneurons and FGNs andgt andgt and andgt respectively The purity of differentiated populations allows us to confidently attribute assay readouts to intrinsic properties of the cellular models and experimental manipulations We have done high fidelity RNAseq of the transcriptome of SCZ FGNs enabled by high purity iPSC differentiation To assess changes in expressions of the dysregulated genes when treated with hit compounds we adapt the RNA mediated oligonucleotide Annealing Selection and Ligation with next gen sequencing RASLseq technology Our team has optimized assay conditions and we can quantitatively profile the expression level of genes in each well of the well plate With our work to date we don t anticipate technical difficulties achieving the following aims Aim Design and test probes for dysregulated genes Based on RNAseq of SCZ FGNs pathway analyses and functionality assessment we chose upregulated and downregulated genes to profile Aim Validate assay with FGNs derived from SCZ patient specific iPSC lines We will quantify the expression of the genes plus house keeping gene controls in FGNs derived from SCZ iPSC lines and compare to available RNAseq data Aim Validate assay with hits from synaptic density primary screen with NINDS Custom Collection which consists of compounds known to influence brain activity FDA approved Assess if hits ameliorate expression dysregulation in DISC FGNs and if hits do not dysregulate expressions in isogenic normal control FGNs We will further assess if the impact on gene expression is consistent with their respective MOAs therapeutic and side effects PROJECT NARRATIVE HIGH THROUGHPUT MULTIPLEXED ASSAY FOR PROFILING GENE EXPRESSIONS OF SCHIZOPHRENIA IPSC DERIVED NEURONS Schizophrenia affects of the population is challenging to treat and is one of the leading public health problems in the US and worldwide of Schizophrenia patients do not respond to treatment and another only partially respond Therapeutics that can treat negative symptoms are lacking JuvoBio Pharmaceuticals has develop innovative stem cell based high throughput assays and will use them to discovery new therapeutic compounds schizophrenia