Transcriptomic analysis of miAtxn2 knockdown effects on lumbar spinal cord in the TAR4/4 sporadic ALS mouse model

Amyotrophic lateral sclerosis (ALS) is characterized by motor neuron death due to nuclear loss and cytoplasmic aggregation of the splice factor TDP-43. Pathologic TDP-43 associates with stress granules (SGs) and downregulating the SG-associated protein Ataxin-2 (Atxn2) using antisense oligonucleotides (ASOs) prolongs survival in the TAR4/4 sporadic ALS mouse model, a strategy that failed in clinical trials, likely due to inadequate target engagement. Here, we used an AAV with potent motor neuron transduction to deliver optimized Atxn2-targeting miRNAs. Atxn2 knockdown was achieved throughout the central nervous system (CNS) at a dose 40x lower than used in other work. In TAR4/4 mice, miAtxn2 treatment increased mean (54%) and median (45%) survival and robustly improved strength-related measures. There was a corresponding reduction of lower motor neuron death as well as reduced inflammatory markers in both motor cortex and spinal cord, and phosphorylated TDP-43 was reduced to wildtype levels. Transcriptome-wide dysregulation in the TAR4/4 model recapitulated ALS-associated gene signatures that were rescued after Atxn2 reduction, identifying therapeutic mechanisms and potential biomarkers for translation. Additionally, in slow progressing hemizygous mice, treatment slowed disease progression and in ALS patient-derived lower motor neurons, our vector transduced >95% of cells at an MOI 4 logs lower than previously reported for AAV and reduced human ATXN2 in a dose-dependent manner. Cumulatively these data support the utility of AAV-mediated RNAi against Atxn2 as a robust and translatable treatment strategy for sporadic ALS.