Antisense oligonucleotide–mediated modulation of SLC6A15 in neurons

Background

In an era of advancing personalized care for the management of specific brain conditions, the SLC6A15 gene has emerged as a candidate for stress vulnerability. This gene, primarily expressed in neurons, encodes a transporter crucial for the uptake of neutral amino acids. As a transmembrane neutral amino acid transporter, it supports amino acid homeostasis and influences glutamatergic neurotransmission [1]. Meanwhile, genome-wide studies have identified risk variants in this gene associated with common psychiatric disorders. In humans, reduced SLC6A15 expression correlates with elevated cortisol, smaller hippocampal volume, heightened stress susceptibility, and reduced cognitive performance [2], whereas knockout mice show neuroprotection against excitotoxicity and enhanced resilience [3]. To probe how reduced SLC6A15 contributes to psychiatric phenotypes, we used antisense oligonucleotides (ASOs)—a proven therapeutic modality with limited application in psychiatry [4]—to modulate SLC6A15 in a 2D human neuronal model derived from induced pluripotent stem cells (hiPSCs).

Methods

Pseudobulk analysis of previously published human orbitofrontal cortex single-nuclei RNA-seq (snRNA-seq) data [5] was used to assess disorder-specific, cell-type SLC6A15 expression and informed model selection. Five ASOs (ASO126, ASO77, ASO175, ASO318, ASO435) targeting the three principal human transcripts (NM_182767.6, NM_018057.7, NM_001146335.3) and a scrambled control were synthesized by Integrated Technologies (IDT). NGN2-differentiated glutamatergic neurons were treated via gymnosis at four concentrations (0.02, 0.2, 2, and 20 μM) on days 41 and 45 of differentiation; cells were harvested on day 49. SLC6A15 mRNA levels were quantified by real-time quantitative polymerase chain reaction (RT-qPCR). Nonlinear regression on normalized data generated dose–response curves and half maximal inhibitory concentration (IC50) estimates using Graphpad prism 10 application. The most potent ASOs were prioritized for follow-up testing.

Results

From the snRNA-seq dataset, SLC6A15 expression was enriched in excitatory and inhibitory neurons and oligodendrocytes, irrespective of disorder status, relative to glia. All ASOs produced concentration-dependent knockdown of SLC6A15 mRNA, achieving maximal inhibition of approximately 90%. A concentration of 0.2 μM emerged as the minimal effective dose, optimizing resolution of ASO potency while reducing the likelihood of off-target effects. Two ASOs—ASO318 and ASO175—were prioritized based on efficacy. Dose–response modeling estimated IC50 values of 0.1245 μM and 0.1430 μM, respectively, with excellent curve fits (R² = 0.9977 and 0.9814). In confirmatory RT-qPCR experiments, both ASOs significantly reduced SLC6A15 mRNA relative to scrambled and vehicle controls (adjusted p < 0.0001). Interestingly, canonical neuronal marker gene (MAP2) and excitatory marker gene (SLC17A7) remain unchanged across treatment conditions.

Conclusions

We provide the first demonstration of in vitro modulation of SLC6A15 using ASOs in human iPSC-derived neurons. These findings validate ASO-mediated knockdown as a potent, target-selective approach for interrogating SLC6A15 function and its context-specific roles in stress susceptibility and resilience. Ongoing studies include protein-level validation and electrophysiological characterization to define the synaptic and functional consequences of SLC6A15 reduction. This work establishes a platform for ASO-based strategies to explore and potentially modulate stress-vulnerability pathways relevant to psychiatric disorders, with implications for both in vitro disease modeling and future therapeutic development.

References

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