Next-generation SOD1 ASOs more potently reduce mRNA and protein in rodent models compared with previous ASOs. Based on an in vitro screen of over 2,000 ASOs targeting the human SOD1 gene and subsequent optimization, 2 particularly potent ASOs targeting the 3′ UTR were selected for further testing (Figure 1A). In a human neuroblastoma cell line (SH-SY5Y), these new ASOs (ASO 1 and ASO 2) potently lowered SOD1 mRNA in a dose-dependent manner (Figure 1B). The new ASOs were more efficacious than the previous SOD1 ASO, 333611 (Figure 1B and Supplemental Figure 1A; supplemental material available online with this article; https://doi.org/10.1172/JCI99081DS1). To determine the potency of these compounds in vivo, ASOs were delivered to the CSF of mice and rats expressing human SOD1G93A via intraventricular (mice) or intrathecal (rats) bolus injection. CSF administration distributes ASOs throughout the brain and spinal cord (8–13). Human SOD1 mRNA was reduced in spinal cords of SOD1G93A mice in a dose-dependent manner (Figure 1C) with approximately 75% maximum reduction of SOD1 following a single injection of ASO 1 or ASO 2. Again, ASO 1 and ASO 2 were substantially more active and efficacious than the previous ASO 333611. Similar dose-dependent reductions in human SOD1 mRNA were achieved in SOD1G93A rats (Figure 1D), with ASO 1 and ASO 2 exhibiting greater activity than 333611 (Figure 1D). Potency of the lead ASO 1 was indistinguishable in SOD1G93A mice and rats, with a lumbar cord EC 50 of 0.9 μg/g and 1.4 μg/g tissue, respectively (Figure 1E). ASO 1 and ASO 2 also exhibit prolonged duration of action, with nearly 10 weeks of sustained SOD1 mRNA lowering following a single intrathecal bolus injection in SOD1G93A rats (Figure 1F). Similarly, misfolded SOD1 also exhibits sustained reduction after a single bolus injection (Figure 1G). Consistent with our previous findings, native SOD1 mRNA (Supplemental Figure 1B) and protein (Figure 1H) are also reduced in spinal cord tissue following ASO treatment.

Figure 1 New SOD1 ASOs reduce mRNA and protein in vitro and in vivo, and are more potent than the previous SOD1 lead. (A) Oligonucleotides containing phosphorothioate backbone modifications (unmodified phosphodiester linkages noted with red o) and 2′-O-methoxyethylribose (MOE; orange) and (S)-2′,4′-constrained 2′-O-ethyl (cEt; blue) groups in the 5′ and 3′ wings were targeted to the 3′ UTR of SOD1 mRNA (ASO 1 and ASO 2), exon 1 of SOD1 mRNA (333611), or nothing in the human or rodent genome (inactive ASO). The sequences evaluated and location of chemical modifications are provided. (B) SHSY5Y cells were treated with SOD1 ASOs by electroporation. After 24 hours, SOD1 mRNA was measured. New SOD1 ASOs are more potent than the previous lead ASO (n = 2 per concentration, average ± range). (C and D) Candidate ASOs for human SOD1 were screened in mice (C) and rats (D) expressing human SOD1. ASOs were injected i.c.v. (mice) and intrathecally (rats) and mRNA was measured in lumbar spinal cord 2 weeks after dosing. Potent ASOs were identified that lowered SOD1 mRNA in both species with ED 50 between 50 μg and 70 μg (n = 3 per dose, individual animals). (E) Tissue concentrations of ASO were measured and correlated with SOD1 mRNA lowering, demonstrating an EC 50 of 0.9 μg/g in mice and 1.4 μg/g in rats (n = 24). (F) A single intrathecal bolus of 500 μg ASO 1 or ASO 2 was given to SOD1G93A rats. SOD1 mRNA levels were assessed in the spinal cord at 2, 4, 8, and 16 weeks after bolus. ASO 1 and ASO 2 suppressed SOD1 mRNA levels for more than 8 weeks (n = 2–7 per time point, average ± SEM). (G) A single intraventricular bolus of ASO 1 or ASO 2 was given to SOD1G93A rats. Misfolded SOD1 protein from spinal cord was assessed at 1, 2, 4, and 8 weeks after bolus (n = 7 per time point, average ± SEM). (H) Six weeks after a single intraventricular bolus of ASO 1, SOD1 protein was quantified in the lumbar spinal cord of SOD1G93A rats. *** P = 0.0005.

Treatment with SOD1-lowering ASO significantly delays disease onset and extends survival in SOD1G93A mice and rats. Expression of mutant SOD1 in SOD1G93A rodent models is known to cause severe atrophy of the limbs and trunk that leads to loss of motor function and eventually death (17). To investigate whether a SOD1-lowering ASO strategy could delay disease parameters, SOD1G93A mice were injected intraventricularly with bolus doses of 300 μg ASO 1 at 50 and 94 days of age. Weight and performance on rotarod were tested weekly. Mice that received ASO 1 maintained weight 26 days longer and performed better on rotarod than mice injected with a control inactive ASO (inactive ASO) at similar concentrations (Figure 2, A and B). Median survival for ASO 1–treated mice was 37 days longer compared with inactive ASO, representing a 22% extension of survival in the mouse model (Figure 2C).

Figure 2 SOD1 ASOs prolong onset, survival, and motor performance of SOD1G93A mutant animals. (A–C) Mice were dosed i.c.v. twice, at day 50 and again at day 94, each time with 300 μg ASO 1 (n = 20 per treatment group, all females). (A) Onset of disease was scored as percentage of animals losing 10% of peak body weight. Median onset for mice treated with the control ASO was 140 days, whereas treatment with ASO 1 increased median onset to 183 days (P < 0.0001, log-rank Mantel-Cox). (B) Motor performance was tested through the rotarod test: mice were tested once a week starting at 80 days of age until they could not stay on the rod for at least 30 seconds. ASO 1 treatment significantly increased rotarod performance: in the control group, the median age at which 50% decrease in performance was reached was 147 days, whereas in the ASO 1–treated animals, the median increased significantly to 182 days (P < 0.0001, 2-way ANOVA). (C) The median survival of mice treated with the control ASO was 168 days, whereas the treatment with ASO 1 increased median survival to 205 days (P < 0.0001, log-rank Mantel-Cox). (D and E) Rats were injected intrathecally with a 1,000 μg single bolus dose of inactive control ASO (n = 16), aCSF vehicle control (n = 19), ASO 333611 (n = 17), ASO 1 (n = 19), or ASO 2 (n = 18). (D) Rats treated with ASO 1 or ASO 2 maintained weight 70 days (P < 0.0001) and 67 days (P < 0.001) longer, respectively, than rats treated with aCSF. 333611 delayed onset of weight loss modestly (median 139 days, compared with aCSF median 121 days). (E) Survival was markedly prolonged in the ASO 1 and ASO 2 treatment groups by 53 days (P < 0.0001) and 64 days (P < 0.0001), respectively, compared with aCSF control treatment in which rats survived to a median age of 166 days. This represents a 32% (ASO 1) and 39% (ASO 2) extension of survival. P values were determined by log-rank (Mantel-Cox) test.

SOD1G93A transgenic rats were injected intrathecally with a single bolus dose of artificial CSF (aCSF) or 1,000 μg ASO vehicle, ASO 333611, ASO 1, ASO 2, or inactive ASO. ASO 333611 is the previous SOD1 ASO as described in reference 8 and used in a phase I study in SOD1 ALS participants (9). Rats assigned to the lead ASO treatment groups performed better in all categories when compared with the vehicle treatment group. SOD1G93A rats treated with ASO 1 or ASO 2 maintained weight 70 days (P < 0.0001) and 67 days (P < 0.001) longer, respectively, than rats treated with aCSF (Figure 2D). In contrast, the intrathecal bolus of ASO 333611 or inactive ASO did not significantly delay onset when compared with the aCSF group (delayed by 17.5 days and 7 days, respectively). Survival was markedly prolonged in the ASO 1 and ASO 2 treatment groups by 53 days (P < 0.0001) and 64 days (P < 0.0001), respectively (Figure 2E), compared with aCSF control treatment in which rats survived to a median age of 166 days. This represents a 32% (ASO 1) and 39% (ASO 2) extension of survival. The effect was similar for both male and female rats (Supplemental Figure 2). This result is substantially improved compared with published results using ASO 333611 infused intraventricularly (10 days, ref. 8) or compared with ASO 333611 delivered intrathecally in this study (195-day median survival, 29-day prolongation) (Figure 2E).

To assess if timing of administration impacts disease onset or survival, SOD1G93A mice were dosed with either ASO 1 or inactive control by i.c.v. bolus at a single time point, either day 80 or 110. Disease onset was delayed and survival markedly extended regardless of the time point of injection (Supplemental Figure 3). There was no significant difference in the onset or survival between the 80-day active ASO treatment and the 110-day active ASO treatment.

SOD1 ASO treatment preserves compound muscle action potential, maintains neuromuscular innervation, and results in lower levels of phospho-neurofilament in SOD1G93A mice. To test whether SOD1 reduction by ASO could affect markers of disease, SOD1G93A mice were injected intraventricularly once at 5 weeks of age with 100 μg ASO 1 and evaluated for changes in compound muscle action potential (CMAP), neuromuscular junction innervation, and serum phospho-neurofilament heavy chain (pNFH) levels. In SOD1G93A mice, CMAP declines over time, preceding the loss of motor neurons (18, 19). SOD1G93A mice treated with ASO 1 at 5 weeks maintained CMAP over the next 12 weeks, whereas the control-treated animals’ CMAP was reduced by more than half over the same time period (Figure 3A). Consistent with CMAP electrophysiological demonstration of preserved muscle function, ASO 1–treated mice maintained innervation of the tibialis anterior muscles in the hind limbs, whereas control-treated animals showed evidence of denervation of more than 75% of muscle endplates (Figure 3B). pNFH increases in CSF and serum in ALS rodent models, human patients with ALS, and other neurodegenerative diseases and it has been proposed as a potential pharmacodynamics marker (20–22). SOD1 mice treated with ASO 1 showed lower levels of pNFH compared with SOD1 mice treated with control ASO (Figure 3C).

Figure 3 One single i.c.v. injection of SOD1 ASO extensively preserves neuromuscular synapses and neuronal loss in SOD1G93A mutant mice. (A–C) Mice were injected once at 5 weeks of age with a single dose of 300 μg ASO 1. Compound muscle action potential (CMAP), neuromuscular junctions (NMJ), and phospho-neurofilament heavy chain (pNFH) were measured at multiple time points (n = 12 per group; average ± SEM). (A) Results validated a significant effect of ASO 1 as compared with the inactive ASO (significant at weeks 7–17). CMAP values recorded from ASO 1–treated animals were not significantly different from those recorded from WT animals except at weeks 15 and 17 (P < 0.001 and P < 0.0001, respectively, 2-way ANOVA). (B) ASO 1 significantly protected SOD1 mice from NMJ loss at the tibialis anterior muscle. Although there was a significant difference in NMJ number between WT animals and SOD1 animals injected with a control ASO at all ages analyzed (data not shown), ASO 1–treated mice did not show significant differences compared with WT mice (P < 0.0001). (C) Blood was collected at baseline (5 weeks) before i.c.v. injection, and then again at 8 and 10 weeks of age. pNFH serum levels were significantly decreased at 8 and 10 weeks of age by ASO 1 treatment (P < 0.001, 2-way ANOVA).

Reversal of SOD1-mediated neuronal dysfunction may be achieved by treatment with SOD1 ASO and is measurable by CMAP and pNFH levels. Two major questions regarding markers of disease in neurodegenerative diseases are: to what extent therapeutics may be able reverse disease and whether this reversal can be measured. SOD1G93A mice were treated at 9 weeks of age when CMAP had clearly begun to decline and pNFH had begun to rise (Figure 4, A and B). Treatment with ASO 3, an SOD1 ASO with dose-dependent reductions in SOD1 mRNA similar to those in ASO 1 and ASO 2 (Supplemental Figure 4), demonstrated a sustained increase in CMAP whereas control ASO–treated mice showed a continued steady decline (Figure 4A). Similarly, pNFH, which at time of injection (9 weeks) had already begun to rise in SOD1G93A mice (Figure 3C), was still significantly lower 8 weeks after treatment with ASO 3 (inactive ASO–treated group, 22 ng/ml ± 1.9 ng/ml; ASO 3–treated group, 12 ng/ml ± 1.2 ng/ml; mean ± SEM, P < 0.001) (Figure 4B). We also found that both pNFH serum and CSF levels were responsive to SOD1 ASO treatment in ALS rats (Supplemental Figure 5). Furthermore, it was recently demonstrated that motor neuron–enriched miR-218 is temporally increased in ALS rat model CSF and its levels correlate with motor neuron loss (23). Here, we found that miR-218 CSF levels were also responsive to SOD1 ASO therapy (Supplemental Figure 5). These results suggest that SOD1-mediated neuronal dysfunction is, at least in part, reversible with a potent therapeutic.

Figure 4 SOD1 ASO injected at 9 weeks of age is able to reverse CMAP amplitudes and lower serum pNFH levels in SOD1G93A mutant mice. (A and B) Mice were injected once at 9 weeks of age with either a control ASO or with ASO 3 (100 μg). (A) CMAP amplitudes at the tibialis anterior muscles were recorded at baseline (5 weeks) and then every other week thereafter (n = 12 per group, average ± SEM). On the week of dosing (9 weeks), CMAP was recorded prior to i.c.v. injection. Typically, SOD1 mice show a steady decline in the CMAP amplitudes recorded at the tibialis anterior; however, one single dose of ASO 3 at 9 weeks of age was able to reverse the trend and by 15 weeks the ASO 3–treated mice had CMAP amplitudes significantly higher than mice treated with a control ASO (P < 0.001, 2-way ANOVA). (B) Blood was collected from each animal at 9, 11, 13, 15, and 17 weeks of age and pNFH levels were quantified. pNFH serum levels of control mice showed a steady increase whereas those of ASO 3–treated mice did not. Levels at 15 weeks: inactive ASO, 19.9 ng/ml ± 5.1 ng/ml; ASO 3, 10.6 ng/ml ± 2.5 ng/ml. Levels at 17 weeks: inactive ASO, 33.96 ng/ml ± 9.3 ng/ml; ASO 3, 16.5 ng/ml ± 4.3 ng/ml. P < 0.0001, 2-way ANOVA, n = 12 per group, average ± SEM.

SOD1 ASOs lower SOD1 mRNA and protein in nonhuman primates. To evaluate the distribution and pharmacokinetic properties of the new SOD1 ASO in a larger brain, ASO 1 was delivered via intrathecal injection to cynomolgus monkeys. ASO 1 was optimized for targeting human SOD1, but it is complementary to nonhuman primate (NHP) SOD1 with a 1-base mismatch, and it is active in NHP cells (Supplemental Figure 6). ASO 1 was evaluated in NHPs at doses of 4 mg, 12 mg, and 35 mg delivered intrathecally, with each monkey receiving 5 doses. One week after the final dose, SOD1 mRNA was lowered broadly in the CNS in a dose-dependent manner (Figure 5A). Quantification of drug levels in various tissues confirmed a pharmacokinetic/pharmacodynamic relationship, with an estimated composite EC 50 of 20 μg/g tissue (Figure 5B). This is consistent across all tissues assessed (Supplemental Figure 7). The decrease in potency in NHP SOD1 compared with human SOD1 in transgenic rodents (Figure 1E, EC 50 0.9–1.4 μg/g) is likely attributable, in part, to the single mismatch of ASO 1 to the NHP SOD1 transcript. SOD1 protein levels in the tissue were lowered by approximately 50% (Figure 5C). A corresponding dose-dependent decrease in CSF SOD1 levels was also observed, confirming CSF SOD1 levels reflect target reduction in the CNS in the larger NHP brain (Figure 5D). CSF SOD1 protein remains reduced approximately 100 days after the last administration (Figure 5E). These data in NHPs demonstrate effectiveness of the SOD1 ASO in a larger animal and are an important step toward understanding the use of the compound in human clinical trials.