A single administration of morpholino antisense oligomer rescues spinal muscular atrophy in mouse

Abstract

Spinal muscular atrophy (SMA) is an autosomal-recessive disorder characterized by α-motor neuron loss in the spinal cord anterior horn. SMA results from deletion or mutation of the Survival Motor Neuron 1 gene (SMN1) and retention of SMN2. A single nucleotide difference between SMN1 and SMN2 results in exclusion of exon 7 from the majority of SMN2 transcripts, leading to decreased SMN protein levels and development of SMA. A series of splice enhancers and silencers regulate incorporation of SMN2 exon 7; these splice motifs can be blocked with antisense oligomers (ASOs) to alter SMN2 transcript splicing. We have evaluated a morpholino (MO) oligomer against ISS-N1 [HSMN2Ex7D(-10,-29)], and delivered this MO to postnatal day 0 (P0) SMA pups (Smn-/-, SMN2+/+, SMNΔ7+/+) by intracerebroventricular (ICV) injection. Survival was increased markedly from 15 days to >100 days. Delayed CNS MO injection has moderate efficacy, and delayed peripheral injection has mild survival advantage, suggesting that early CNS ASO administration is essential for SMA therapy consideration. ICV treatment increased full-length SMN2 transcript as well as SMN protein in neural tissue, but only minimally in peripheral tissue. Interval analysis shows a decrease in alternative splice modification over time. We suggest that CNS increases of SMN will have a major impact on SMA, and an early increase of the SMN level results in correction of motor phenotypes. Finally, the early introduction by intrathecal delivery of MO oligomers is a potential treatment for SMA patients.

Figures

Figure 1.

Illustration of SMN2 exon and intron 7 with highlighted ISS-N1 and the target site for morpholino HSMNEx7D(−10–29) (MO).

Figure 2.

Analysis of exon 7 incorporation in SMN2 transcript. Smn+/−; SMN2+/+; Δ7SMN+/+ mice were injected by P0 ICV with low- (27 μg), middle- (54 μg) or high- dose (81 μg) MO. RNA from brain (A) and spinal cord (B) tissue was isolated and cDNA amplified with SMN2-specific primers. All dosings show increased full-length SMN2 (top band) versus exon 7-deficient SMN2 (bottom band) at all time points (P7, P21, P45, P65) relative to scMO-injected control animals (HiC = 16 copies SMN2 (8SMN2+/+; Smn−/−); WT, wild type. (C) ddPCR for relative full-length SMN2 (FL-SMN2) relative to cyclophilin; low-, middle-, high-dose MO (dose–response curve); P7 brain and spinal cord tissue. (D) Quantitative RT–PCR for full-length SMN2 in spinal cord P7–P65 (low, middle, high dose). There is increased P7 full-length SMN2, with decay at later time points.

Figure 3.

Central versus peripheral SMN2 splice modulation. (A) RT–PCR systemic analysis of full-length SMN2 after P0 MO ICV injection. There is no increased SMN2 exon 7 incorporation in visceral structures (the heart, liver, kidney) when compared with spinal cord (n= 2). (B) ddPCR (full-length SMN2 relative to cyclophilin) of the brain, spinal cord, heart and liver after P0 MO ICV (54 µg) or P0 FV (50 µg/g) injection in Smn+/−; SMN2+/+; Δ7SMN+/+ mice. Results per organ are relative to scramble-injected control, which has been set at 1 on the y-axis. There is increased CNS splice modulation after both ICV and peripheral dosing. There is no increased splicing in the heart and liver after ICV injection, suggesting limited translocation across the CSF–blood barrier or rapid systemic degradation. (C) ddPCR (FL-SMN2 relative to cyclophilin) of the brain, spinal cord, heart and liver after P0 MO ICV (54 µg) injection in Smn−/−; SMN2+/+; Δ7SMN+/+ mice; SMA scramble-injected control per each organ. Results are displayed as absolute relative ratios on the y-axis. There are large increases in CNS splice modulation, and a more modest increase in liver full-length SMN2.

Figure 4.

Analysis of SMN induction after MO delivery. Smn+/−; SMN2+/+; Δ7SMN+/+ mice were injected by P0 ICV with low- (27 μg), middle- (54 μg) or high-dose (81 μg) MO. Quantification of SMN by human specific antibody relative to actin in the brain (A) and spinal cord (B) for each dose at P7, P21, P45 and P65. All doses increased P7 SMN (strongest increase with high dose) and decay through P65 (n= 3 for each dose per time point). (C and D) Representative western blots for human SMN and actin in the brain (A) and spinal cord (B) after low-dose injection.

Figure 5.

Motor neuron SMN expression increases in MO-injected Δ7SMN SMA mice. (A–C) 54 μg MO (middle) dose was injected by P0 ICV. Lumbar spinal cord tissue was harvested at P7, sectioned and stained with human specific anti-SMN KH antibody. (A) Motor neurons in the ventral horn were identified by HB9:GFP transgenic expression. (B) SMN is found in the cytoplasm and in gems in the nucleus of motor neurons, as well as other cell types, throughout the spinal cord. Insert highlights the motor neuron indicated by the arrowhead. (C) The merged image of SMN expression (red) in the motor neuron (green). Insert highlights the motor neuron indicated by the arrowhead. (D–F) SMN expression in a non-injected carrier control animal. (D) Motor neurons identified by HB9:GFP expression. (B) A low level of SMN expression from SMN2 is present throughout the spinal cord. (C) The merged image of SMN expression (red) in the motor neuron (green). Scale bar = 200 µm.

Figure 6.

Survival and weight gain are increased in MO-treated Δ7SMN SMA animals. (A) The Kaplan–Meier survival curve for low (n= 8), middle (n= 10) and high dose (n= 13). The median survival for low (83 ± 37.7 days, maximum 161), middle (104 ± 15.0 days, maximum 148) and high dose (112 ± 6.6 days, maximum 153) were not statistically different (log-rank P> 0.8 for all pairwise comparisons). The median survival for each group was significantly extended when compared with scMO (6 mm, 81 µg)-injected Δ7SMN SMA animals (15 ± 1.1 days, log-rank P< 0.001 for all pairwise comparisons). (B) MO-treated Δ7SMN SMA mice increased mass nearly parallel to that of control animals (Smn+/+; SMN2+/+; SMNΔ7+/+, scMO injection) through ∼P30, at which point the mass curves leveled while control animals continued to gain weight. P80 mean: low dose = 16 ± 0.9 g, middle dose: 17 ± 0.6 g, high dose: 19 ± 1.9 g, control = 22 ± 1.4 g. (C) The Kaplan–Meier survival curve after 5 µg (n= 4) and 10 µg (n= 3) MO ICV P0 injection (censored, through P30).

Figure 7.

Treated SMA animals have an improved motor phenotype. (A) Righting response quickly approaches 100% by P3 in ICV MO-treated animals, while scMO-injected SMNΔ7 SMA animals have a rapid decline in righting ability during the first week of life. No SMA animals injected with scMO were able to right after P6. (B) Mass-corrected forelimb and hindlimb grip strength is equivalent between control animals (Smn+/+; SMN2+/+; SMNΔ7+/+, scMO injection) and MO-treated SMNΔ7 SMA animals (grip strength assessed at P30 in Newtons/mg body mass; forelimb: control 34.5 ± 2.2 versus SMA 34.2 ± 2.1, hindlimb: control 28.2 ± 5.0 versus SMA 25.8 ± 4.4).

Figure 8.

Alternative MO injection strategies do not increase the survival beyond P0 ICV MO injection. Three cohorts of mice each received an injection of MO by P0 FV (50 µg/g body mass, n= 3), P0 FV and ICV (50 µg/g peripheral, 54 µg ICV, n= 5) or P0 and P30 ICV (with stereotactic guidance, 54 µg/g P0, 18 µg/g P30, n= 4). Survival for all groups was equivalent to high-dose P0 ICV MO injection (log-rank P> 0.09).

Figure 9.

Delayed ICV injection yields intermediate weight gain and survival. (A) SMNΔ7 SMA mice treated at P4 with 54 μg MO/g body mass (‘high dose’ equivalent) had increased weight gain when compared with SMNΔ7 SMA controls, but was less than the weight gain displayed by P0 MO-treated SMNΔ7 SMA animals [P30 mean 10.6 ± 1.5 g (P4) versus 13.6 ± 1.0 g (P0)]. (B) P4 ICV MO-injected animal survival was also increased (median 41 ± 14.2 days, maximum 78 days) when compared with scMO-injected animals (15 ± 1.1 days; log-rank P< 0.001), but was decreased when compared with SMNΔ7 SMA animals treated at P0 with high-dose MO (112 ± 6.6 days, maximum 153 days, log-rank P< 0.01). (C) SMNΔ7 SMA mice were injected with P4 MO by FV. Survival was modestly extended when compared with scMO-injected animals, and there was a decreased survival when compared with animals injected by P4 ICV MO (median 21 ± 0.354 days, log-rank P< 0.014).