AAV9-DARS2 Gene Therapy Rescues Phenotype in Leukoencephalopathy with Brainstem and Spinal Cord Involvement and Lactate Elevation Patient Cells and Neuronal Dars2 Deficient Mice

Objective: Leukoencephalopathy with brainstem and spinal cord involvement and lactate elevation (LBSL) is a rare, autosomal recessive disorder caused by variants in the gene DARS2. DARS2 is an essential and ubiquitously expressed enzyme that catalyzes the attachment of aspartate to its cognate tRNA for mitochondrial protein translation. LBSL is clinically characterized by progressive spasticity, ataxia, and dorsal column dysfunction, and is considered a primary axonopathy with secondary demyelination. Methods: Herein, we tested the efficacy of gene supplementation, using adeno-associated virus, serotype 9 (AAV9)-DARS2 in LBSL patient cells, as well as in an LBSL mouse phenolog in which Dars2 was deleted in CamKIIα-expressing neurons of the hippocampus and cortex. Results: In vitro, patient neurons treated with AAV9-DARS2 showed increased gene expression of the gene mirrored by improved mitochondrial function, axonal growth, and reduced lactate release, despite variation in impairment across lines. Knockout mice showed improved behavior and reduced cortical neurodegeneration 6 months after a single intracerebroventricular injection of AAV9-DARS2. Interpretation: Together, this work provides proof-of-concept data that gene supplementation can improve cell function and survival for an extended period of time. AAV9 therapy has proven especially useful for loss of function monogenetic disorders, and these data may support further investigation into therapies for LBSL. ANN NEUROL 2026;99:59–72.