Abstracts

This is a Late Breaking abstract

Rationale: The full realization of next generation CNS therapeutics will require the input of vast, high quality data sets directly linked to fundamental human biology with a deeper understanding of phenotypes resulting from pathological perturbations at the level of individual neurons. Here, we report on a platform for the characterization of human cellular models which provides deep neuronal insights for selecting optimal therapeutic candidates. Q-State’s BRITE^TM (Bioengineered neuRonal Insight-driven Therapeutic Engine)system was applied in a human induced pluripotent stem (iPS) cell-derived neuronal model of Tuberous Sclerosis (TSC) to establish a functional phenotype that could be used to screen for novel chemical matter and target mechanisms. TSC is a rare genetic disorder caused by mutations in the mTOR regulators TSC1 and TSC2. The majority of TSC patients (75-90%) present with seizures during infancy and many suffer with intractable epilepsy throughout life, underscoring the need for novel therapeutics._x000D_
Methods: Given that loss-of-function mutations in TSC2 cause TSC, we used CRISPR/Cas9 gene editing tools to create a collection of TSC2^+/+, TSC2^+/- and TSC2^-/- isogenic iPS cell lines, which were then converted into cortical excitatory neurons using transcriptional programming. Cultures of human differentiated neurons were then characterized using Q-State’s BRITE^TM platform, which included assessment of morphological (high-content imaging), molecular (RNA-Seq signatures) and functional (optical electrophysiology) phenotypes. In parallel, we generated iPS cell lines and neurons from a cohort of TSC2^+/- patients and controls and used these reagents for validation studies._x000D_
Results: High-content imaging analyses revealed increase in soma size and upregulation of pS6 kinase in TSC2^-/- neurons, which are well-established readouts of disruption of TSC2 function and mTOR signaling. RNA-Seq studies identified >4,000 genes that were differentially expressed in TSC2^-/- neurons and a significant fraction (>50%) of this molecular signature could be rescued with mTOR modulators. A robust and high throughput screening (HTS)-compatible (Z’ >0.4) functional phenotype was established in the isogenic neuronal models and validated in the patient-derived neurons. This multiparametric excitability phenotype, which included changes in spike timing and spike shape features, was used to complete a complex (chronic and multi-intervention treatments) primary screening of ~30,000 small molecule compounds which led to the identification and validation of >500 hits with functional rescue of the disease phenotype. Compound analogues have been tested for the most interesting hits along with eADME, which led to the identification of at least 3 hit compound Series._x000D_
Conclusions: Q-State’s platform was successfully used to complete a high throughput, multiparametric functional phenotypic screen in a human loss-of-TSC2-function model of TSC, providing multiple hits which may be explored by medicinal chemistry. _x000D_
Funding: None