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. 2018 Aug 1;128(8):3558-3567.
doi: 10.1172/JCI99081. Epub 2018 Jul 16.

Antisense oligonucleotides extend survival and reverse decrement in muscle response in ALS models

Alex McCampbell  1 , Tracy Cole  2 , Amy J Wegener  3 , Giulio S Tomassy  1 , Amy Setnicka  3 , Brandon J Farley  1 , Kathleen M Schoch  3 , Mariah L Hoye  3 , Mark Shabsovich  3 , Linhong Sun  1 , Yi Luo  1 , Mingdi Zhang  1 , Nicole Comfort  1 , Bin Wang  1 , Jessica Amacker  1 , Sai Thankamony  1 , David W Salzman  1 , Merit Cudkowicz  4 , Danielle L Graham  1 , C Frank Bennett  2 , Holly B Kordasiewicz  2 , Eric E Swayze  2 , Timothy M Miller  3
Affiliations

Antisense oligonucleotides extend survival and reverse decrement in muscle response in ALS models

Alex McCampbell et al. J Clin Invest. .

Abstract

Mutations in superoxide dismutase 1 (SOD1) are responsible for 20% of familial ALS. Given the gain of toxic function in this dominantly inherited disease, lowering SOD1 mRNA and protein is predicted to provide therapeutic benefit. An early generation antisense oligonucleotide (ASO) targeting SOD1 was identified and tested in a phase I human clinical trial, based on modest protection in animal models of SOD1 ALS. Although the clinical trial provided encouraging safety data, the drug was not advanced because there was progress in designing other, more potent ASOs for CNS application. We have developed next-generation SOD1 ASOs that more potently reduce SOD1 mRNA and protein and extend survival by more than 50 days in SOD1G93A rats and by almost 40 days in SOD1G93A mice. We demonstrated that the initial loss of compound muscle action potential in SOD1G93A mice is reversed after a single dose of SOD1 ASO. Furthermore, increases in serum phospho-neurofilament heavy chain levels, a promising biomarker for ALS, are stopped by SOD1 ASO therapy. These results define a highly potent, new SOD1 ASO ready for human clinical trial and suggest that at least some components of muscle response can be reversed by therapy.

Keywords: ALS; Genetic diseases; Neurodegeneration; Neuroscience; Therapeutics.

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Conflict of interest statement

Conflict of interest: AM, GST, BJF, LS, YL, MZ, ST, DLG, and DWS are employees of Biogen. TC, CFB, HBK, and EES are employees of Ionis Pharmaceuticals. Ionis Pharmaceuticals provides antisense oligonucleotides to TMM. Biogen provides support to TMM for clinical studies. TMM has participated in a medical advisory board for Biogen and is a consultant for Cytokinetics. TMM and Washington University have licensed technology regarding SOD1 protein kinetic measurements to C2N. MC is a consultant for Cytokinetics, Lilly, Orion, Biohaven, Wave and Biogen.

Figures

Figure 1
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 ED50 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 EC50 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.
Figure 2
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.
Figure 3
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).
Figure 4
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.
Figure 5
Figure 5. SOD1 ASO lowers SOD1 in nonhuman primate CNS and CSF.
(A–C) The safety of the ASO was assessed in cynomolgus monkeys, which also provided an opportunity to understand the on-target activity of the ASO and the utility of SOD1 in the cerebrospinal fluid as a marker of target suppression in the tissue. Monkeys were dosed with artificial CSF or 4, 12, or 35 mg ASO 1, with each monkey receiving 5 doses. One week after the final dose, SOD1 mRNA and protein were quantified and associated with ASO concentration across brain regions. (A) A dose-dependent lowering of mRNA across CNS regions, including the lumbar spinal cord, was observed (n = 6, average ± SEM). L, lumbar; Th, thoracic; Cerv, cervical; Fr, frontal cortex; Motor, motor cortex; Hip, hippocampus; Pons, pons; Cb, cerebellum. (B) A pharmacokinetic/pharmacodynamic relationship could be observed, with an estimated IC50 of 20 μg/g tissue (n = 126, each point representing one tissue). (C) A dose-dependent lowering of SOD1 protein across CNS regions, including the lumbar spinal cord, was observed (n = 6, average ± SEM). aCSF, artificial CSF. (D) A dose-dependent lowering of SOD1 protein in CSF as compared with the vehicle-treated group was observed (n = 6; line, average; 4 mg, P = 0.0019; 12 mg, P = 0.0006; 35 mg, P = 0.0003; 1-way ANOVA). **P < 0.005; ***P < 0.001. (E) CSF SOD1 protein remains significantly reduced as compared with day 0, roughly 100 days following the last intrathecal ASO administration (n = 6, average ± SEM; ****P = 0.0001; 1-way ANOVA).
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