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Figure S1. Splicing effect of control ASO, therapeutic ASO-10-27 or ASO-20-37. (A) ICV injection of 100 μg or IP injection of 200 mg/kg/day control ASO did not affect SMN2 splicing in spinal cord or liver, respectively, analysed at PS7 or PS14 (n = 3). (B) Time-course analysis of the effect of ICV injection of 100 μg ASO-10-27 on SMN2 splicing in spinal cord (n = 3). (C) Compared with PS7, 10 or 25 μg ASO-20-37 further inhibited exon 7 inclusion at PS30 in a dose-dependent manner (n = 3). Twenty-five micrograms ASO has a stronger effect than 10 μg. (D) Similar splicing effect in PS7 spinal cord between ICV injection of 25 μg ASO-20-37 with or without additional IP injection of 200 mg/kg/day ASO-20-37 (n = 3). FL, Δ7 and Δ5 + Δ7: full-length, exon-7-skipped transcripts and mRNA lacking both exon 5 and exon 7, respectively. S: 5 μl saline, C: 100 μg control ASO; 25 + IP: combination of ICV injection of 25 μg and IP injection of 200 mg/kg/day ASO-20-37.

Figure S2. ASO uptake in tissues 7 days (A) or 30 days (B) after ICV injection of 100 μg ASO-20-37 (IHC). ASO is broadly detected in CNS cells, and mainly in liver sinusoids. ASO is only weakly detectable in heart, lung and quadriceps muscle. Compared with PS7, PS30 uptake declines in the CNS. The brain and spinal cord regions indicated by open circles are also displayed at higher magnification in the insets. Scale bar: 1000 and 200 μm for 6 and 40× magnification. Saline: ICV injection of 5 μl saline.

Figure S3. Phenotypic effect of ASO-20-37. (A) Mice that receive ICV injection of 25 μg ASO-20-37 are smaller and show kyphosis. (B) Mice begin to lose body weight around 20 days after IP injection of 200 mg/kg/day ASO-20-37. Control mice (n = 10) and mice injected with ASO-20-37 (n = 12) were analysed. (C) IP injection of ASO-20-37 had no effect on motor function, analysed by grip strength (n = 7. n.s., p = 0.9122) and rotarod task (n = 7. n.s., p = 0.3599) measured at PS21. (D) ICV injection of 10 μg ASO-20-37 had no effect on motor function, analysed by grip strength (n = 3. n.s., p = 0.6601) and rotarod task (n = 3. n.s., p = 0.4227) measured at PS120. Control: ICV injection of 100 μg control ASO; IP control: IP injection of 200 mg/kg/day control ASO. We analysed data using two-tailed t tests.

Figure S4. Muscle histology. No overt muscle pathology in quadriceps (top) or tibialis anterior (bottom) is seen at end-stage PS70 in the mice given ICV injection of 25 μg ASO-20-37. H&E, acetate non-specific esterase and NADH dehydrogenase staining. Scale bar: 200 μm.

Figure S5. Amelioration by systemically administered therapeutic ASO. Two hundred milligrams per kilogram per day therapeutic ASO-10-27 was injected intraperitoneally 14 and 15 days after IP injection of 200 mg/kg/day ASO-20-37. (A) Extended survival by ASO-10-27. Control mice (n = 10), mice without therapy (n = 15) and with IP injection of ASO-10-27 (n = 10) were analysed. (B) Retained body weight. Control mice (n = 10), mice without therapy (n = 12) and with IP injection of ASO-10-27 (n = 10) were analysed. The median survival days after the first injection of ASO-20-37 are given in parentheses for each group. (C) Increased exon 7 inclusion in SMN2 in P30 liver (n = 3). (D) Ameliorated histological change in liver at PS30. There was only mild hepatocellular necrosis with sporadic enlarged cells (arrow), and inflammatory cell infiltration (arrowhead), particularly neutrophils. H&E staining. Scale bar: 200 μm. (E) Retained serum AST and ALT levels (n = 5. n.s., p = 0.7732 and 0.4442, respectively) and IGF1 levels (n = 5. n.s., p = 0.1472). (F) Retained mRNA levels of hepatic Igf1 and Igfals at PS30 (n = 3). For Igfals in the mice treated with ASO-20-37 only, the lower band in each doublet was quantitated. IP control or IP200 ASO-20-37: IP injection of 200 mg/kg/day control ASO or ASO-20-37 for 2 days, respectively; +PS14/15 IP therapy: IP injection of 200 mg/kg/day ASO-20-37 for 2 days and IP injections of 200 mg/kg/day ASO-10-27 at PS14 and PS15. We analysed data using two-tailed t tests, except for the logrank test for survival analysis.

Table S1. Sequences of the uniform 2′-MOE ASOs with phosphorothioate backbone and 5-methylcytosines used in this study.

Table S2. Echocardiographic measurements before and 25 days after IP injection of control ASO or ASO-20-37.

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