Antisense correction of SMN2 splicing in the CNS rescues necrosis in a type III SMA mouse model

Abstract

Increasing survival of motor neuron 2, centromeric (SMN2) exon 7 inclusion to express more full-length SMN protein in motor neurons is a promising approach to treat spinal muscular atrophy (SMA), a genetic neurodegenerative disease. Previously, we identified a potent 2'-O-(2-methoxyethyl) (MOE) phosphorothioate-modified antisense oligonucleotide (ASO) that blocks an SMN2 intronic splicing silencer element and efficiently promotes exon 7 inclusion in transgenic mouse peripheral tissues after systemic administration. Here we address its efficacy in the spinal cord--a prerequisite for disease treatment--and its ability to rescue a mild SMA mouse model that develops tail and ear necrosis, resembling the distal tissue necrosis reported in some SMA infants. Using a micro-osmotic pump, we directly infused the ASO into a lateral cerebral ventricle in adult mice expressing a human SMN2 transgene; the ASO gave a robust and long-lasting increase in SMN2 exon 7 inclusion measured at both the mRNA and protein levels in spinal cord motor neurons. A single embryonic or neonatal intracerebroventricular ASO injection strikingly rescued the tail and ear necrosis in SMA mice. We conclude that this MOE ASO is a promising drug candidate for SMA therapy, and, more generally, that ASOs can be used to efficiently redirect alternative splicing of target genes in the CNS.

Figure 1.

Dose response study of ASO 10–27 in the spinal cord after ICV infusion in adult mice. ASO 10–27 at the indicated daily doses was infused for 7 d into the right cerebral lateral ventricle of type III SMA heterozygous mice. On day 8, mice were euthanized. (A) Thoracic spinal cord RNA samples were analyzed by radioactive RT–PCR. FL and Δ7 represent full-length and exon 7-skipped SMN2 transcripts, respectively; “% incl” is the percentage of the FL spliced mRNA in total SMN2 transcripts. Four representative samples are shown for each dose. (B) Quantitation of the data in A plus unpublished data (n = 5) (Y Hua and AR Krainer, unpubl.). Error bars represent standard deviations (SD). (C) Quantitation by real-time RT–PCR. Both the FL mRNA (+exon 7) and exon 7-skipped mRNA (−exon 7) (n = 5) were normalized with GAPDH, and the data were further normalized to the level of the corresponding transcript in the saline-treated controls (dose, 0 μg per day). (D) Thoracic spinal cord protein samples obtained from four heterozygotes (the same mice as in A) and four additional homozygotes, treated with either saline or 50 μg per day of ASO, were analyzed by Western blotting with antibodies SMN-KH (hSMN) or SMN-BD (total SMN); β-tubulin was used as a loading control. Histograms on the right show the protein quantitation.

Figure 2.

Detection of ASO 10–27 and hSMN protein in spinal cord by immunostaining after ICV infusion. (A) Thoracic spinal cord samples isolated 1 d after 7-d ICV infusion of saline or ASO 10–27 at the indicated daily doses were analyzed by immunohistochemistry with a pAb that specifically recognizes the phosphorothioate backbone in ASOs, or with SMN-KH mAb specific for human SMN. Nuclei were counterstained with DAPI. (B) Confocal immunofluorescence images of thoracic spinal cord double-labeled with SMN-KH mouse mAb and anti-ChAT rabbit pAb, followed by incubation with goat Alexa fluor 488 anti-mouse and Alexa fluor 568 anti-rabbit secondary antibodies. Bars, 25 μm.

Figure 3.

RT–PCR analysis of SMN2 splicing in different regions of the spinal cord, and duration of action of ICV-infused ASO 10–27. (A) Total RNA samples isolated from the cervical (C), thoracic (T), or lumbar (L) spinal cord of mice treated by 7-d ICV infusion with the indicated daily doses of ASO 10–27 or a six-mismatch control ASO were analyzed by radioactive RT–PCR as in Figure 1. Representative triplicate samples out of four mice for each dose are shown. (B) After a 7-d ICV infusion of type III SMA model heterozygous mice at 50 μg per day, total RNA samples were prepared from thoracic spinal cord isolated at the indicated time points (0 indicates 1 d after ICV infusion) and analyzed by radioactive RT–PCR. (Con) Saline-treated controls. Representative triplicate samples from four to five mice for each time point are shown. (C) Quantitation of SMN2 splicing in different regions of the spinal cord. An asterisk (*) indicates a significantly lower percentage of exon 7 inclusion in the lumbar than in the cervical region (P = 0.045, n = 4, two-tailed paired Student's t-test) and thoracic regions (P = 0.031, n = 4). (D) Quantitation of the duration-of-action data (n = 4-5). For all of the time points, the difference from the control was highly significant (P ≤ 9.3 × 10⁻⁹, two-tailed Student's t-test). Error bars, SD.

Figure 4.

2′-OMe but not MOE ASO induces an increase in Aif1 expression, a marker of microglial activation. The MOE ASO (MOE), a six-mismatch ASO (Mismatch), and the 2′-OMe ASO (2′-OMe) (for sequences, see the Materials and Methods) were delivered into the CNS of heterozygous type III SMA mice by ICV infusion for 7 d at the indicated doses (n = 5 for all doses, except for the 2′-OMe treatment at 100 μg per day, for which two mice died prematurely). On day 9, mice were euthanized, and the whole brain (A) and lumbar spinal cord (B) were collected for total RNA extraction. Aif1 mRNA levels were analyzed by real-time RT–PCR with specific primers and normalized to GAPDH mRNA. The data were further normalized to the saline controls (dose, 0 μg per day, white bars). MOE ASO gave no increase of Aif1 expression in the brain, and a 1.2-fold increase at only the highest dose (100 μg per day) in the lumbar spinal cord (P = 0.01). 2′-OMe ASO caused a large increase in Aif1 mRNA levels at doses ≥10 μg per day in the spinal cord, and at doses of 100 μg per day in the brain. Error bars represent standard deviations. (*) P < 0.05; (**) P < 0.01; (***) P < 0.001, compared with the saline controls.

Figure 5.

Dose response study of ASO 10–27 in the lumbar spinal cord after a single embryonic ICV injection. Using a glass micropipette, 2 μL of ASO 10–27 was injected into a cerebral lateral ventricle of individual embryos at approximately E15 (Smn^+/− or Smn^−/−; SMN2^+/+) at the indicated doses. (A) The effect of ASO 10–27 on SMN2 splicing was assayed at P7. Total RNA isolated from the lumbar spinal cord was analyzed by radioactive RT–PCR as in Figure 1. Three representative samples from four to five treated mice are shown. (B) Quantitation of the dose response radioactive RT–PCR data from lumbar spinal cords obtained at P7 or P30 (n = 4–5). For all of the doses, the difference between P7 and P30 was significant (P ≤ 0.02, two-tailed Student's t-test). (C) Quantitation of the effects of ASO 10–27 in different spinal cord regions in P7 neonates; two doses (5 and 10 μg) were chosen for comparison. An asterisk (*) indicates a significantly lower percentage of exon 7 inclusion in the lumbar spinal cord than in the cervical (P = 0.017, n = 4, two-tailed paired Student's t-test) and thoracic (P = 0.016, n = 4) regions.

Figure 6.

Phenotypic rescue of type III SMA mice (Smn^−/−; SMN2^+/+) after a single embryonic ICV injection of ASO 10–27. (A) Tail lengths were measured from P7 to 12 wk old, at weekly intervals. E15 embryos were injected with saline vehicle (n = 10) or 10 μg (n = 17) (ASO-10) or 20 μg (n = 11) (ASO-20) of ASO 10–27 in a volume of 2 μL. Normal mice (Smn^+/+ or Smn^+/−) were used as positive controls (n = 10). (B) Radioactive RT–PCR of tail RNA samples obtained from mice treated with saline or 20 μg of ASO 10–27 at approximately E15. (C) Representative 2-mo-old mice to show ASO rescue of tail and ear necrosis. The two mice on the left have normal tails and ears (the one at the far left is Smn^+/−; SMN2^+/+, and the other one is Smn^+/+; SMN2^+/+), the two mice in the middle are type III SMA mice (Smn^−/−; SMN2^+/+) that were treated as embryos with 20 μg of ASO 10–27, and the two SMA mice (Smn^−/−; SMN2^+/+) on the right received no treatment. The enlargements show the right ears of one treated and one untreated mouse.