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

Period of Performance: 07/15/2016 - 01/14/2017


Phase 1 SBIR

Recipient Firm

Juvobio Pharmaceuticals, Inc.
WESTON, MA 02493
Principal Investigator


Project Summary - High throughput multiplexed assay for profiling gene expressions of schizophreniaiPSC-derived neurons This proposal is to develop and validate a secondary high-throughput screening (HTS) assay for newtherapeutics for schizophrenia (SCZ). By assessing primary hit compounds? ability to ameliorate transcriptionaldysregulation in SCZ neurons and not to dysregulate normal neurons in 384-well format, the potentialmechanism 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 avalidated primary HTS synaptic density assay using forebrain glutamatergic neurons (FGNs) derived frominduced pluripotent stem cells (iPSCs) bearing a Disrupted in schizophrenia 1 (DISC1) frameshift mutation.This proposed secondary screen, our primary screen and other confirmatory assays we have developed form apowerful toolset for SCZ drug discovery. SCZ, a major public health problem, affects around 1% of the world?s population. It manifests withdiverse symptoms, is challenging to treat, has complex genetic risk associations and poorly understoodetiology. 30% of patients do not respond to medications, 30% show partial response, and only 30% enter fullremission. SCZ medications tend to be partially effective for positive symptoms but have limited or no effectfor negative symptoms or cognitive impairments, which are the most important predictors for long-term socialfunctioning. Hence, new psychotropic therapeutics are need to address significant unmet medical needs. We have optimized differentiation protocols that produce highly pure populations of cortical neuralprogenitors (NPCs), GABAergic interneurons and FGNs (>99%, >80% and >90% respectively). The purity ofdifferentiated populations allows us to confidently attribute assay readouts to intrinsic properties of the cellularmodels and experimental manipulations. We have done high-fidelity RNAseq of the transcriptome of SCZ FGNs, enabled by high-purity iPSCdifferentiation. 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 theexpression level of 200-300 genes in each well of the 384-well plate. With our work-to-date, we don?tanticipate technical difficulties achieving the following aims:Aim 1. Design and test probes for 400 dysregulated genes. Based on RNAseq of SCZ FGNs, pathwayanalyses and functionality assessment, we chose 200 upregulated and 200 downregulated genes to profile.Aim 2. Validate assay with FGNs derived from 15 SCZ-patient specific iPSC lines. We will quantify theexpression of the 400 genes plus house-keeping gene controls in FGNs derived from 15 SCZ iPSC lines, andcompare to available RNAseq data.Aim 3. Validate assay with hits from synaptic density primary screen with NINDS Custom Collection 2,which consists of 1040 compounds (known to influence brain activity, 75% FDA approved). Assess if hitsameliorate expression dysregulation in DISC1 FGNs and if hits do not dysregulate expressions in isogenicnormal control FGNs. We will further assess if the impact on gene expression is consistent with theirrespective MOAs, therapeutic and side effects.