Using Induced Pluripotent Stem Cells Derived from Patient Blood for Transcriptional Modeling and Drug Repositioning in Phelan-Mcdermid Syndrome

Background: Autism spectrum disorder (ASD) has high heritability and a prevalence of nearly 1% worldwide, but heterogeneity of patients has made identifying the underlying etiology difficult. By focusing on monogenic disorders with high penetrance for causing ASD, common pathways might be identified. Phelan-McDermid syndrome (PMS) is one such monogenic ASD-associated syndrome that is caused by haploinsufficiency of the gene SHANK3, which encodes for a scaffolding protein of the post-synaptic density at glutamatergic synapses. While animal models provide great insight into the pathways involved in PMS, many features of the disease may not be captured because of brain variation across species. One approach to deal with this shortcoming is to generate induced pluripotent stem cells (iPSCs) from patients that can then be differentiated into neural progenitor cells (NPCs) and neurons. Researchers from other groups using iPSC-derived neurons from PMS patients have found excitatory synaptic deficits similar to those seen in animal models.

Objectives: NPCs and neurons derived from patient and sibling iPSCs illuminate two distinct time points in the neurodevelopmental trajectory of PMS and allow for the identification of disrupted pathways that can be targeted through drug repositioning. With this project, we aim to 1) generate high-quality iPSC clones from blood samples collected from PMS patients and their unaffected siblings; 2) differentiate these iPSCs into both NPCs and neurons to capture the neurodevelopmental profile of PMS; 3) identify PMS-associated differential gene expression in iPSC-derived neural cells by RNA sequencing; 4) integrate the results with other PMS and ASD models to identify convergent findings; and 5) identify candidate drugs by comparing gene expression patterns for FDA-approved drugs with PMS-associated expression.

Methods: Blood samples from 6 patient/sibling pairs were collected and reprogrammed using a modified, non-integrating Sendai virus protocol. For each individual, 2-3 iPSC clones were validated and banked before using them for monolayer-based NPC generation, followed by 6 weeks of terminal differentiation into neurons. NPC and neuron samples were validated with immunolabeling for established NPC and neuron markers before subjecting them to RNA sequencing and differential expression. A transcriptional signature for PMS was generated and compared with expression data from a SHANK3 rat model from our lab to identify convergent pathways. For drug discovery, compounds with known expression profiles were ranked by their anticorrelation to the PMS signature.

Results: Differentially expressed genes between case and control lines were identified for each cell type and used for gene ontology enrichment analysis to identify the most significantly disrupted pathways. Comparing the iPSC-derived neural cells with a SHANK3 knockout rat model of PMS from our lab revealed convergent dysregulation of Wnt signaling and extracellular matrix-related genes, while the top candidates from drug repositioning endeavors were enhancers of GABAergic signaling.

Conclusions: Elucidating the transcriptional profile of neural cells derived from PMS patient iPSCs provides both a unique perspective on the neurobiological underpinnings of PMS, and a template that can be used with known drug expression profiles for repositioning and screening.