LAUSR

Spinal muscular atrophy (SMA), the leading genetic cause of infant mortality, arises from survival motor neuron (SMN) protein insufficiency resulting from SMN1 loss. Approved therapies circumvent endogenous SMN regulation and require repeated dosing or may wane. We describe genome editing of SMN2, an insufficient copy of SMN1 harboring a C6>T mutation, to permanently restore SMN protein levels and rescue SMA phenotypes. We used nucleases or base editors to modify five SMN2 regulatory regions. Base editing converted SMN2 T6>C, restoring SMN protein levels to wild type. Adeno-associated virus serotype 9–mediated base editor delivery in Δ7SMA mice yielded 87% average T6>C conversion, improved motor function, and extended average life span, which was enhanced by one-time base editor and nusinersen coadministration (111 versus 17 days untreated). These findings demonstrate the potential of a one-time base editing treatment for SMA. Description Base editing in a single treatment Spinal muscular atrophy is the leading genetic cause of infant death. It arises from the lack of a protein called survival motor neuron (SMN). Drugs that increase SMN are effective but require repeated dosing or may fade over time. Arbab et al. identified genome-editing strategies that permanently correct SMN protein levels to normal levels by converting a partially active gene encoding SMN into a fully active form. In a mouse model, treatment with base editors that efficiently and precisely make this change increased life span and rescued motor function. A one-time combination treatment of a base editor and a current spinal muscular atrophy drug further improved outcomes in mice. —DJ Base editing enables a one-time treatment for spinal muscular atrophy that rescues disease pathology and extends life span in mice. INTRODUCTION Spinal muscular atrophy (SMA) is the leading genetic cause of infant mortality. SMA results from survival motor neuron (SMN) protein insufficiency after homozygous loss of the SMN1 gene. A closely related gene, SMN2, differs from SMN1 by a C6T substitution (i.e., a C-to-T transition at position 6) in exon 7 that results in a truncated SMNΔ7 protein that fails to fully compensate for SMN1 loss. Two recently approved SMA drugs partially restore SMN protein levels through splice isoform switching. A third drug uses viral gene complementation to restore SMN levels. Although up-regulation of SMN levels by these approved drugs effectively treats SMA, current therapies circumvent endogenous regulation of SMN, do not fully restore SMN levels, and either require repeated dosing or may fade over time. A one-time, permanent treatment that restores endogenous gene expression and preserves native SMN regulation may address these limitations of existing SMA therapies. RATIONALE Genome editing of SMN2, which is present in all SMA patients, could enable a one-time treatment for SMA that restores normal SMN transcript and protein levels while preserving their endogenous regulatory mechanisms. We developed one-time genome editing approaches targeting endogenous SMN2 that restore SMN protein abundance to normal levels and rescue disease phenotypes in cell and mouse models of SMA. We tested 79 base editing and nuclease strategies that modify five posttranscriptional and posttranslational regulatory regions in SMN2 to increase SMN protein levels. RESULTS Each of the SMN2 nuclease and base editing strategies tested durably increased SMN protein levels between 9- and 50-fold. Base editing efficiently converted SMN2 to SMN1 genes and, unlike nuclease editing strategies or current SMA drugs, fully restored SMN transcript and protein levels to those of wild-type cells (~40-fold increase) with minimal off-target editing across the genome and transcriptome. Intracerebroventricular injection of adeno-associated virus serotype 9 encoding an adenine base editor (AAV9-ABE) resulted in 87% average conversion of SMN2 C6T among transduced cells in the central nervous system of Δ7SMA mice, improved motor function, and extended life span, despite Δ7SMA mice having a much shorter window for treatment than human patients (≤6 days for mice versus months to years for humans) that ends earlier than typical in vivo base editing time scales (weeks). One-time in vivo coadministration of AAV9-ABE with the antisense oligonucleotide drug nusinersen expanded the therapeutic window for gene correction, further improving the life span of AAV9-ABE–treated animals to an average of 111 days, compared with an average of 17 days for untreated animals. CONCLUSION Despite the incongruent timeline of base editing–mediated rescue for ideal rescue of Δ7SMA mice, AAV9-ABE treatment yielded substantial improvements in life span and motor function. Combination treatment with nusinersen enables Δ7SMA mouse rescue that resembles presymptomatic up-regulation of SMN levels. In humans, the therapeutic window is much longer. Therefore, we anticipate that AAV9-ABE may achieve presymptomatic rescue as a standalone therapeutic in SMA patients. Our study also demonstrates the compatibility of base editing with nusinersen, which may inform future clinical applications. Together, these findings support the potential of base editing as a future one-time treatment for SMA that restores native SMN production while preserving endogenous regulatory mechanisms of SMN expression. Base editing of SMN2 rescues SMA in mice. (A) A customized ABE converts insufficient SMN2 genes into healthy SMN1 genes to produce full-length SMN protein. (B) Dual-AAV9–mediated delivery of ABE and green fluorescent protein (GFP) into SMA neonates. (C) In vivo conversion of SMN2 C6T in the central nervous system of treated animals. (D) Motor unit number estimation (MUNE) in SMA mouse muscle after base editing treatment. het, heterozygous. (E) Survival of SMA mice after base editing treatment. ns, not significant. *P ≤ 0.02, ***P ≤ 0.005, ****P ≤ 0.001.