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

Background: 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. Methods: 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. Results: AI-based stability predictors uniformly indicated destabilization of GAT-1(A305V) and GAT-1(A305T). A305V reduced Conclusions: 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.