Abstracts

SLC6A1-related disorder is a severe developmental epileptic encephalopathy with an urgent need for an effective disease-modifying therapy. It is caused by loss of function in GAT1, a primary GABA transporter in the brain, leading to impaired GABA clearance and widespread neural circuit dysfunction. Endogenous GAT1 neuronal expression is restricted to inhibitory neurons, where it plays a crucial role in synaptic and extra-synaptic GABA uptake. AAV-mediated gene delivery of SLC6A1 has the potential to provide a one-time curative treatment for this disease, yet the importance of targeting expression to defined brain cell types remains unknown. This study aims to determine the significance of cell-type specificity in gene therapy for SLC6A1-related disorder.

We developed AAV-based therapeutics to express human SLC6A1 either selectively in inhibitory neurons or in all neurons to test whether cell-type selectivity is essential for the safe rescue of phenotypes. AAV vectors were delivered via retro-orbital injection into a mouse model of SLC6A1-related disorder at a juvenile age (postnatal day 21). Therapeutic efficacy was assessed through multifaceted analyses, including hippocampal synaptosome GABA uptake assays, acute slice electrophysiology recording, chronic video EEG/EMG recordings, and behavioral tests assessing motor function and short-term learning and memory.

Inhibitory neuron-specific expression of SLC6A1 was well tolerated in diseased mice and led to significant improvements across major disease-relevant phenotypes. Treated mice showed restored GABA uptake function in hippocampal synaptosomes, normalized kinetics of evoked inhibitory postsynaptic currents, and reduced epileptiform activities, including decreased spike-and-wave discharges (absence seizures), spike trains, and interictal spikes, and restored FFT features. Behavioral testing revealed rescue of failure-to-thrive phenotype, tremor, hindlimb clasping, rotarod performance, and short-term learning deficits in contextual and cued-induced fear conditioning. In contrast, pan-neuronal expression of SLC6A1 led to increased mortality, convulsive seizures, and limited rescue of disease phenotypes.

Our findings demonstrate that targeting SLC6A1 expression specifically to inhibitory neurons is necessary and sufficient for safe and efficacious gene therapy in a mouse model of SLC6A1-related disorder. The inhibitory-specific strategy improves a wide range of phenotypes in juvenile mice, including epileptiform activity, motor dysfunction, and cognitive deficits. Pan-neuronal expression, while partially effective, introduces significant safety concerns. These results support inhibitory neuron-selective gene therapy as a promising approach for SLC6A1-related disorder.

This project was supported through the Paul G. Allen family foundation’s continued investment in the Allen Institute and through a sponsored research agreement with BioMarin Pharmaceutical for basic scientific discovery research.