Targeting ASO-10-27 oligonucleotides to the mouse CNS. ASO-10-27 was efficiently targeted to motor neurons of the spinal cord of neonatal mice 14 days after intracerebroventricular injection. Double immunohistochemical staining was performed for the phosphorothioate backbone of the oligo-nucleotide (first column) and mouse choline acetyltransferase (ChAT; second column). (A to D) Colocalization signals (third column) are shown for the cervical (B), thoracic (C), and lumbar (D) regions of the spinal cord after ASO-10-27 treatment; control saline-treated mice did not show colocalization (A). Scale bar, 100 μm.

ASO-10-27 treatment increases myofiber size in SMA mice. (A to D) H&E staining of the quadriceps (left column) and intercostal (right column) muscles of untreated SMA mice (A), ASO-mismatch–treated SMA mice (B), ASO-10-27–treated SMA mice (C), and untreated WT mice (D) at 16 days. (E) The average myofiber cross-sectional area for each group was quantified. The P values between the different treatment groups were determined by one-way ANOVA and Bonferroni multiple post hoc comparisons (P < 0.05; P < 0.001). Data are means ± SEM. Scale bar, 50 μm.

ASO-10-27 treatment improves function and survival of SMA mice. (A to F) Results of the body weight (A and B), righting reflex(C and D), gripstrength (E), and hindlimb splay (F) at 8 days (A and C) and 16 days (B, D, E, and F). (G) A single administration of different doses (0.5 to 8 μg) of ASO-10-27 was injected into P0 mice with severe SMA, and their survival was plotted on a Kaplan-Meier curve. The median survival was 18 to 26 days, which was a significant improvement compared to untreated SMA mice (0.5 μg, P = 0.0237; 1 μg, P = 0.0007; 2 μg, P = 0.0001; 4 μg, P < 0.0001; 8.0 μg, P = 0.0004). In contrast, SMA mice treated with ASO-mismatched oligonucleotides did not show an increase in survival (4 μg, P = 0.5081). (H) Percent weight gains between P14 and P7 with the different doses of ASO-10-27 in SMA mice. SMN, untreated SMA mice (n = 18); MM, SMA mice treated with 4 μg of ASO-mismatched oligonucleotide (n = 14); SMA mice treated with 0.5 μg (n = 10), 1 μg (n = 9), 2 μg (n = 10), 4 μg (n = 20), or 8 μg (n = 13) of ASO-10-27. The P values between the different treatment groups in (A) to (F) were determined by one-way ANOVA and Bonferroni multiple post hoc comparisons (P < 0.05; P < 0.01; P < 0.001). The P values in the survival curve were analyzed with the Mantel-Haenszel test by comparing each treatment group to the untreated SMA group. Data are means ± SEM.

ASO-10-27 treatment boosts SMN protein expression in SMA mice. Treatment with ASO-10-27 increased exon 7 inclusion during SMN2 splicing, and increased SMN protein expression and the numbers of motor neurons in the spinal cord after intracerebroventricular injection of neonatal mice with severe SMA at postnatal day 0 (P0). (A) The amount of SMN2 mRNA that contained exon 7 relative to SMN2 mRNA in untreated SMA mice was determined by reverse transcription–polymerase chain reaction (RT-PCR) at 16 days. (B) The amount of SMN protein relative to that in untreated wild-type (WT) mice was determined by Western blotting at 16 days. (C) For the pharmacodynamic/pharmacokinetic analysis, the absolute ASO tissue concentration in the cervical region was determined by capillary gel electrophoresis; the amount of SMN protein relative to that in untreated WT mice in the thoracic region was determined by Western blotting; and the amount of SMN2 mRNA that contained exon 7 relative to that in untreated SMA mice in the lumbar region was determined by RT-PCR at 3, 16, and 30 days. (D) The number of motor neurons in the cervical, thoracic, and lumbar regions was determined by ChAT immuno-staining at 16 days. SMA, untreated SMA mice; MM, ASO-mismatch–treated SMA mice; 10-27, ASO-10-27–treated SMA mice; WT, untreated WT mice. The P values between the different treatment groups were determined by one-way ANOVA and Bonferroni multiple post hoc comparisons (P < 0.01; P < 0.001). Data are means ± SEM.

ASO-10-27 treatment improves neuromuscular junction structure in SMA mice. (A to D) In toto staining of muscle fibers from the quadriceps (left column) and intercostal (right column) muscles of untreated SMA mice (A), ASO-mismatch–treated SMA mice (B), ASO-10-27–treated SMA mice (C), and untreated WT mice (D) at 16 days. The axons of motor neurons at the presynaptic termini (green) were labeled with antibody to neurofilament medium (NF-M). The acetylcholine receptors of the postsynaptic termini were stained with α-bungarotoxin (α-Bung; red). The overlay of both signals (yellow) shows the innervation of the muscle and nerve at the motor end plate. As exemplified by the neuromuscular junctions in untreated SMA mice, the presynaptic termini displayed a collapsed structure that typifies a pathological neuromuscular junction (A). (E) The percentage of neuromuscular junctions (NMJ) that contained this hallmark pathology was quantified at 16 and 30 to 40 days. The P values between the different treatment groups were determined by one-way ANOVA and Bonferroni multiple post hoc comparisons (P < 0.05; P < 0.01; P < 0.001). Data are means ± SEM. Scale bar, 10 μm.

Therapeutic levels of ASO-10-27 in monkey spinal cord. Intrathecal (IT) and intracerebroventricular (ICV) infusions of ASO-10-27 loaded the monkey spinal cord with therapeutic levels (>8 μg/g) of the oligonucleotide. (A) ASO tissue concentrations in the cervical, thoracic, and lumbar regions after the administration of a 3-mg dose over different infusion times. (B) Immunohistochemical staining for the phosphorothioate backbone of ASO-10-27 showed a punctate intracellular signal in all ASO-10-27–treated groups, but not in monkeys that received saline. The immunopositive signal was detected in both large and small cell bodies, suggesting neuronal and glial uptake of the oligonucleotide. Data are means ± SEM. Scale bar, 100 μm.