Therapeutic rescue of Spinal Muscular Atrophy mouse models by AAV9-Exon Specific U1 snRNA part2

Exon-Specific U1 snRNAs (ExSpeU1) are modified U1 snRNAs that promotedefinition of defective exons. In Spinal Muscular Atrophy (SMA), their transgenicgermline expression rescued the severe pathology in a mouse model. Here, weevaluate the therapeutic levels, the activity and safety profile of ExSpeU1 deliveredby AAV9 vectors. AAV9-ExSpeU1 systemic treatment increases SMN2 exon 7inclusion and SMN protein mainly in peripheral organs and rescues the phenotype inmild and severe SMA mice. In the severe mouse, the treatment improves theneuromuscular function and significantly increases the life span (from 10 to 219days). The rescued adult mice showed normal morphology of the neuromuscularjunction, normal number of motoneurons in spinal cord and a significant persistenceafter one month of the ExSpeU1 expression in the AAV9 transduced tissues. A lowtherapeutic level of ExSpeU1 RNA relative to the endogenous U1snRNA (~ 0.1-0.3%/U1wt) is required for efficacy. RNA-SEQ analysis on two representative tissuesshowed a parallel recover of the SMA-induced expression and splicing profilesmostly related to DNA damage, cell cycle control and acute phase response. Offtarget analysis by RNA-SEQ in a human model cell line that overexpress highamount of ExSpeU1 (~20- 40%/U1wt) did not identify significant differentiallyexpressed genes or splicing events. These results indicate that AAV-mediateddelivery of a modified U1 snRNP particle is a safe and valid therapeutic option inSMA to improve SMN2 defective splicing.