Dual targeting of astrocytic and endothelial GLUT1 enables functional rescue in GLUT1 deficiency syndrome

Glucose transporter 1 deficiency syndrome (GLUT1-DS) is a metabolic encephalopathy caused by impaired glucose transport into the brain. Although GLUT1 in brain microvascular endothelial cells at the blood-brain barrier has been recognized as essential for glucose transport into the brain, the role of astrocytic GLUT1 has remained uncertain, limiting progress in gene therapy for GLUT1-DS. Here we show that astrocytic GLUT1 is broadly expressed in both mouse and human brains and is indispensable for maintaining cerebrospinal fluid glucose, cognitive performance, and motor coordination. Conditional haploinsufficiency in either astrocytes or endothelial cells reproduced GLUT1-DS-like phenotypes, and full rescue required AAV-mediated GLUT1 restoration in both cell types. To this end, we developed AAV-AST, a blood-brain barrier-penetrant vector with strong astrocyte tropism, and identified a regulatory element (Region d) that recapitulates endogenous GLUT1 expression. Together, these findings establish dual targeting of astrocytic and endothelial GLUT1 as a rational therapeutic strategy, paving the way for next-generation therapies for GLUT1-DS and related neuro-metabolic disorders.