Temporal Single Cell RNAseq to Identify Genes and Pathways Affected by 15q11.2 Duplication in Autism iPSC-Derived Differentiating Cortical Neurons

Period of Performance: 08/16/2016 - 08/15/2017


Phase 1 SBIR

Recipient Firm

Juvobio Pharmaceuticals, Inc.
WESTON, MA 02493
Principal Investigator


PROJECT SUMMARY We aim to establish a single-cell RNAseq database of differentiating cortical neuronal progenitors(NPCs) and neurons derived from patient-specific induced pluripotent stem cells (iPSCs) for neuro-developmental diseases (NDDs). The database will also include data on differentiation, morphology, formationand functionality of synapses, annotations of analytical findings, and will be supported by the most advancedsingle-cell RNAseq bioinformatics tools. We will provide all this, together with corresponding NPCs andneurons for research reagents, as tool to support academic research and drug discovery for NDDs in the field. One in 68 children born in 2002 are diagnosed with Autism Spectrum Disorder (ASD), a public healthpriority in the US. Genetic predispositions in ASD are thought to contribute to the primary pathology by alteringneuronal development, evidenced partly by altered gene expressions. Patient-specific induced pluripotentstem cells (iPSCs) have been shown to recapitulate specific disease phenotypes through the neurogenicprocess and can serve as effective disease models. Hence, single-cell RNAseq along the accessible andcontrolled process of differentiating ASD-specific iPSCs into neural progenitors and subtypes of neurons canprovide insights into the temporal and multi-lineage dimensions of ASD pathogenesis and biomarkers fordiagnostics, progression, and therapeutics discovery. Copy-number variants (CNVS) at 15q11.2 is a prominent risk factor for neurological disorders includingASD, epilepsy, and schizophrenia. 15q11-q13 duplication/triplication represent the most common CNVs inpatients with ASD (up to 3%). Duplications and microdeletions can both lead to the same disorders,suggesting the importance of this region in normal neurological functions and the necessity to study the impactof both duplications and deletions for a full understanding of the mechanisms. Phase I utilizes four 15q11.2 duplication iPSC lines from ASD patients and two control iPSC lines to:Aim 1: Characterize neural differentiation of ASD and control iPSC-derived neurons. Differentiate iPSCsinto cortical NPCs, glutamatergic, and GABAergic subtypes. Identify deficits in ASD lines throughmorphological studies and structural analyses of synapses.Aim 2: Generate single-cell RNAseq datasets of differentiating NPCs and neuronal subtypes at ninetime points during differentiation.Aim 3: Perform bioinformatics analyses. Reconstitute the molecular dynamics underlying neuronaldifferentiation. Validate experimental conditions including sampling frequency and number of cells. Identify,validate molecular signatures underlying 15q11.2 duplication?s impact on neuronal differentiation and functions.Phase II: utilize the experimental conditions established here to generate single-cell RNAseq datasets frommultiple ASD iPSC lines that harbor different genetic mutations to be included in the database; build databasestructure and user interface, and seek to identify aberrant differentiation and functional development caused byASD mutations as well as genes and pathways that are commonly and differentially affected across multipleASD mutations. We will devote resources to annotate the database with our own findings and those that arepublished by peers to enhance its utility to subscribers.