Comparison of the efficacy of MOE and PMO modifications of systemic antisense oligonucleotides in a severe SMA mouse model

Abstract

Spinal muscular atrophy (SMA) is a motor neuron disease. Nusinersen, a splice-switching antisense oligonucleotide (ASO), was the first approved drug to treat SMA. Based on prior preclinical studies, both 2'-O-methoxyethyl (MOE) with a phosphorothioate backbone and morpholino with a phosphorodiamidate backbone-with the same or extended target sequence as nusinersen-displayed efficient rescue of SMA mouse models. Here, we compared the therapeutic efficacy of these two modification chemistries in rescue of a severe mouse model using ASO10-29-a 2-nt longer version of nusinersen-via subcutaneous injection. Although both chemistries efficiently corrected SMN2 splicing in various tissues, restored motor function and improved the integrity of neuromuscular junctions, MOE-modified ASO10-29 (MOE10-29) was more efficacious than morpholino-modified ASO10-29 (PMO10-29) at the same molar dose, as seen by longer survival, greater body-weight gain and better preservation of motor neurons. Time-course analysis revealed that MOE10-29 had more persistent effects than PMO10-29. On the other hand, PMO10-29 appears to more readily cross an immature blood-brain barrier following systemic administration, showing more robust initial effects on SMN2 exon 7 inclusion, but less persistence in the central nervous system. We conclude that both modifications can be effective as splice-switching ASOs in the context of SMA and potentially other diseases, and discuss the advantages and disadvantages of each.

Figure 1.

MOE10-29 led to better therapeutic outcomes than PMO10-29 in severe SMA mice. (A) Survival curves after two SC injections of saline (n = 12), MOE10-29 (4 nmol/g, n = 14; 12 nmol/g, n = 14) and PMO10-29 (4 nmol/g, n = 14; 12 nmol/g, n = 14) between P0 and P1. (B) Body-weight curves after SC injections of MOE10-29 and PMO10-29 as in (A). (C) Representative pictures of high-dose MOE10-29- and PMO10-29- treated SMA mice on P60. (D) Tail length was measured weekly for up to 14 weeks (n = 12). (*) P < 0.05 high dose versus low dose of the same ASO; (^#) P < 0.05 versus all other groups.

Figure 2.

SMN2 splicing changes in mouse peripheral tissues after SC injection of MOE10-29 or PMO10-29. Splicing analysis of liver (A and B), heart (C andD), kidney (E andF) and muscle (G andH) tissues from MOE10-29- and PMO10-29-treated mice by radioactive RT-PCR at three time points (P7, P15 and P30) after treatment as in Figure 1 (n = 4). (*) P < 0.05 high dose versus low dose of the same ASO. % incl, percentage of exon 7 inclusion; (^#) P < 0.05 MOE versus PMO at the same dose.

Figure 3.

SMN protein expression in mouse peripheral tissues after SC injection of MOE10-29 or PMO10-29. Western blotting analysis of SMN levels in mouse liver (A andB), heart (C andD), kidney (E andF) and muscle (G andH) tissues at three time points (P7, P15 and P30) after the treatments described in Figure 1 (n = 4). (*) P < 0.05 high dose versus low dose of the same ASO; (^#) P < 0.05 MOE versus PMO at the same dose.

Figure 4.

SMN2 splicing changes in mouse CNS tissues after SC injection of MOE10-29 or PMO10-29. Splicing analysis of spinal cord (A and B) and brain (C and D) from MOE10-29- and PMO10-29-treated mice by radioactive RT-PCR at three time points (P7, P15 and P30) after treatment as in Figure 1 (n = 4). (*) P < 0.05 high dose versus low dose of the same ASO; (^#) P < 0.05 MOE versus PMO at the same dose.

Figure 5.

SMN protein expression in mouse CNS tissues after SC injection of MOE10-29 and PMO10-29. Western blotting analysis of SMN levels in mouse spinal cord (A and B) and brain (C and D) tissues at three time points (P7, P15 and P30) after treatment as in Figure 1 (n = 4). (*) P < 0.05 high dose versus low dose of the same ASO; (^#) P < 0.05 MOE versus PMO at the same dose.

Figure 6.

Gem counts in motor neurons of spinal cord segments L1–L2 obtained from ASO-treated SMA mice. (A) SMA mice were SC injected twice with saline (n = 4), MOE10-29 (4 nmol/g, n = 4; 12 nmol/g, n = 4) and PMO10-29 (4 nmol/g, n = 4; 12 nmol/g, n = 4) between P0 and P1. Tissues were collected on P9. Gems and motor neurons were labeled with SMN and ChAT; nuclei were counterstained with DAPI. (B) Quantitation of gems per 100 motor neurons from (A). (C) Percentages of motor neurons containing 0, 1, 2, 3, or ≥4 gems from A. (*) P < 0.05 high dose versus low dose of the same ASO; (^#) P < 0.05 MOE versus PMO at the same dose.

Figure 7.

Motor-neuron counts in spinal cord segments L1–L2 and motor-function tests of SMA mice. (A) SMA mice were treated with saline (n = 4), MOE10-29 (4 nmol/g, n = 4; 12 nmol/g, n = 4) and PMO10-29 (4 nmol/g, n = 4; 12 nmol/g, n = 4) as described in Figure 6. Untreated heterozygous mice (Het, n = 13) were used as normal controls. Tissues were collected on P9. Motor neurons and nuclei were labeled with ChAT and DAPI. (B) Motor-neuron number was calculated based on (A) (four mice per group and three counts per mouse). (C and D) Grip strength and rotarod tests of rescued SMA mice (n = 12) at 3 months of age. (*) P < 0.05 high dose versus low dose of the same ASO; (#) P < 0.05 MOE versus PMO at the same dose.

Figure 8.

NMJ staining of the flexor digitorum brevis 2 and 3 (FDB-2/3) muscles of SMA mice. (A) SMA mice were treated with saline (n = 4), MOE10-29 (4 nmol/g, n = 4; 12 nmol/g, n = 4) and PMO10-29 (4 nmol/g, n = 4; 12 nmol/g, n = 4) as in Figure 6. Untreated heterozygous mice (Het, n = 4) were used as normal controls. FDB-2/3 were collected on P9 and stained for neurofilament with anti-neurofilament (blue), nerve terminals with anti-synaptophysin (green) and motor endplates with α-bungarotoxin (α-BTX, red). (B) NMJ area was measured from (A) (four mice per group and three counts per mouse). (C) Quantification of perforations identified by α-BTX, based on (A) (4 mice per group and 3 counts per mouse). (D) Percentages of innervated endplates (red), partially denervated endplates (blue), and fully denervated endplates (white) were quantitated based on (A) (four mice per group and three counts per mouse). (*) P < 0.05 high dose versus low dose of the same ASO; (^#) P < 0.05 MOE versus PMO at the same dose.