Fig 2: The 2nd-generation vector was preferentially expressed in the CNS compared to peripheral tissues.(A, B) SMN protein expression analysis. SMA neonates were injected with vectors via the facial vein with a dose of 3.3E + 14 vg/kg on P0. On (A) Day 30 and (B) Day 90, mouse brain, spinal cord, heart, and quadricep muscles were harvested for Western blot analysis. Age-matched healthy carriers were used as a reference. Untreated SMA mice were harvested at two weeks of age as a control. Quantitative analysis is on the right side. SMN expression shown as a ratio to β-tubulin and normalized to healthy carriers. (C) Immunostaining analysis in mouse lumbar spinal cord at 30 days. ChAT (red, a motor neuron marker), SMN (green), and merged (yellow). (D) Immunostaining of mouse lumbar spinal cord at 30 days. NeuN (red, a neuronal marker), SMN (green), and merged (yellow). Data represent the mean ± SD. One-way ANOVA, *P < 0.05; **P < 0.01; ns, not significant. Source data are available online for this figure.

Fig 3: Supraphysiological levels of SMN contributes to hepatoxicity in mice.(A) Schematic representation of the designed plasmids. A vector genome identical to onasemnogene abeparvovec was produced as the benchmark vector. A codon-optimized human SMN1 gene (co-hSMN1) was synthesized and incorporated into a scAAV genome with the cytomegalovirus enhancer/chicken β-actin promoter (CMVen/CB-hSMN1) in the presence or absence of a synthetic intron as Vector 1 and Vector 2, respectively. (B) A representative image of Western blot (n = 3) of Neuro2a cells transfected with the plasmids in (A). (C) Kaplan-Meier survival curves of homozygous SMNΔ7 mice injected at P0 via the facial vein at three different doses (3.3E + 14 vg/kg, n = 12; 1.1E + 14 vg/kg, n = 7; 0.5E + 14 vg/kg, n = 5). The benchmark vector was injected as a reference at a dose of 3.3E + 14 vg/kg (n = 13). Non-injected SMNΔ7 mice (n = 4) were used as controls. (D) Western blot analysis of SMN expression in mouse liver 8 days after injection of Vector 1 and the benchmark vector. (E) Photographs of neonatal mice 8 days after injection. (F) H&E staining was performed to show liver pathology. Bar = 200 μm. (G) Both SMA mice and healthy carriers were injected at P0 with 3.3E + 14 vg/kg of Vector 1 or the benchmark vector. Liver histopathology was analyzed by H&E staining for each animal 8 days post-injection. Animals with damaged livers were counted. (H) The rAAV-cohSMN1 vector construct with reduced SMN expression in liver contains three copies of a liver-specific, miR-122 binding site in the Vector 1 genome and is packaged in AAV9 (top). SMA mice were treated at P0 by facial vein injection at a dose of 3.3E + 14 vg/kg, and at eight days post-injection, mouse livers were harvested for Western blot analysis (bottom). (I) Animal weights were measured daily during the 8-day study period (n = 8 in the PBS group; n = 10 in Vector #3 group and n = 7 in Vector #1 group). Error bars, s.d. (J) Pathological evaluation of liver, by H&E staining was conducted to determine the number of animals with liver damage, and (K) vector genome copy number (n = 5 in each group). Student’s t-test. **P < 0.01. Error bars, s.d. Source data are available online for this figure.

Fig 4: Respiratory function assessment by plethysmography in SMA-treated mice and healthy carriers.On Day 30, respiratory function was assessed by tidal volume, peak inspiratory flow, and respiratory rate. Bars represent the mean and error bars represent SD. One-way ANOVA, *P < 0.05; **P < 0.01; ns, not significant.

Fig 5: Direct Vpr Targets Involved in G2/M Arrest(A) Correlation between depletion of MCM10, MUS81, and EME1 by each Vpr mutant tested in the experiment shown in Figure 2 and the extent of G2/M arrest caused by that mutant. The red line shows linear regression analysis.(B) Example DNA staining showing G2/M arrest caused by shRNA-mediated depletion of MCM10, representative of three independent experiments. Watson pragmatic modeling was used to identify cells in G1 (blue), S (gray), or G2/M (red) phase.(C) Real-time qRT-PCR analysis of MCM10 mRNA abundance in cells transduced with control or MCM10-targeting shRNA. Values were generated using the ΔΔCT method relative to GAPDH mRNA abundance and normalized to the control condition. Bars show mean and SEM of three technical replicates.(D) Targeted shRNA screen of direct Vpr target proteins identified here whose depletion correlated with G2/M arrest in the experiment detailed in Figure 2. Bars show means and SEM of at least two replicates from more than three independent experiments. Dashed lines show control average ± 3 SDs. The control condition contains combined data from three different control shRNAs.(E) Real-time qRT-PCR analysis of mRNA abundance in cells transduced with control or targeting shRNA. Values were generated using the ΔΔCT method relative to GAPDH mRNA abundance and normalized to the control condition. Bars show mean and SEM of three technical replicates.(F) Correlation between depletion of SMN1, CDCA2, and ZNF267 by each Vpr mutant tested in the experiment shown in Figure 2 and the extent of G2/M arrest caused by that mutant.(G) Example DNA staining showing G2/M arrest caused by shRNA-mediated depletion of SMN1, CDCA2, and ZNF267 using a second independent shRNA; representative of at least two independent experiments.