4 Phenylbutyrate Plus Gene augmentation: A Dual Therapy To Rescue of SLC6A1 Variant Associated Developmental And...

Pathogenic variants in SLC6A1, which encodes the γ-aminobutyric acid (GABA) transporter GAT-1, cause developmental and epileptic encephalopathies (DEEs) through reduced GABA uptake, impaired transporter trafficking, and functional haploinsufficiency. 4-Phenylbutyrate (PBA) is a clinically available small molecule with chemical-chaperone and histone-deacetylase-inhibitor activities that can rescue misfolded GABAergic proteins, but variant-level rescue data are needed to guide precision treatment.

We report a newly identified de novo SLC6A1 missense variant, p.Ala305Val (A305V), in a patient with myoclonic-atonic epilepsy and a developmental and epileptic encephalopathy phenotype. A305V was compared with the residue-matched comparator p.Ala305Thr (A305T). Variant effects were evaluated by (i) protein-structure prediction across nine stability-prediction algorithms using the cryo-EM-derived human GAT-1 template (PDB 7Y7W); (ii) 3H-GABA uptake assays in HEK293T cells and in human iPSC-derived astrocytes and cortical neurons; (iii) live-cell confocal microscopy of ER colocalization; (iv) pharmacologic rescue with PBA, TUDCA and salubrinal (v) and GAT-1 cDNA gene-augmentation, alone and in combination with PBA.

AI-based stability predictors uniformly indicated destabilization of GAT-1(A305V) and GAT-1(A305T). A305V reduced ³ H-GABA uptake across HEK293T, astrocyte, and neurons. The mutant transporter accumulated within the endoplasmic reticulum (ER), with ER colocalization rising from approximately 30% in wildtype to ∼80% in A305V; PBA reduced ER retention to approximately ∼40% and restored total GAT-1 fluorescence toward wildtype levels. Pharmacochaperones (PBA, TUDCA) restored GABA uptake for the mutant transporters. Wildtype GAT-1 gene augmentation improved mutant GAT-1 uptake and combined PBA-plus-augmentation produced rescue greater than either intervention alone in the available dose-response ranges.

SLC6A1 A305V is a trafficking-impaired, loss-of-function GAT-1 variant whose dysfunction is tractable to two convergent therapeutic axes: pharmacologic correction of folding and trafficking, and augmentation of functional transporter dose. These findings support a two-pronged precision-medicine framework for SLC6A1-related DEEs in which PBA increased the transporter function augmented by increased gene therapy.

This work links the patient-derived SLC6A1 A305V variant to a defined molecular mechanism-GAT-1 destabilization, ER retention, and reduced GABA uptake-and demonstrates that the deficit is reversible by two independent interventions that converge at the same downstream endpoint of functional surface transporter. Because PBA is already clinically deployable and GAT-1 cDNA augmentation models a future viral or non-viral gene therapy, the combined-rescue logic provides a falsifiable path for precision medicine in SLC6A1-related DEEs: chemical chaperoning corrects the folding bottleneck while transporter augmentation increases the pool available for rescue.