A novel morpholino oligomer targeting ISS-N1 improves rescue of severe spinal muscular atrophy transgenic mice

Abstract

In the search for the most efficacious antisense oligonucleotides (AOs) aimed at inducing SMN2 exon 7 inclusion, we systematically assessed three AOs, PMO25 (-10, -34), PMO18 (-10, -27), and PMO20 (-10, -29), complementary to the SMN2 intron 7 splicing silencer (ISS-N1). PMO25 was the most efficacious in augmenting exon 7 inclusion in vitro in spinal muscular atrophy (SMA) patient fibroblasts and in vitro splicing assays. PMO25 and PMO18 were compared further in a mouse model of severe SMA. After a single intracerebroventricular (ICV) injection in neonatal mice, PMO25 increased the life span of severe SMA mice up to 30-fold, with average survival greater by 3-fold compared with PMO18 at a dose of 20 μg/g and 2-fold at 40 μg/g. Exon 7 inclusion was increased in the CNS but not in peripheral tissues. Systemic delivery of PMO25 at birth achieved a similar outcome and produced increased exon 7 inclusion both in the CNS and peripherally. Systemic administration of a 10-μg/g concentration of PMO25 conjugated to an octaguanidine dendrimer (VMO25) increased the life span only 2-fold in neonatal type I SMA mice, although it prevented tail necrosis in mild SMA mice. Higher doses and ICV injection of VMO25 were associated with toxicity. We conclude that (1) the 25-mer AO is more efficient than the 18-mer and 20-mer in modifying SMN2 splicing in vitro; (2) it is more efficient in prolonging survival in SMA mice; and (3) naked Morpholino oligomers are more efficient and safer than the Vivo-Morpholino and have potential for future SMA clinical applications.

FIG. 1.

Schematic representation of SMN2 exon 7 splicing regulatory elements. The functional elements regulating exon 7 splicing in SMN2 are depicted along the top. Antisense oligonucleotides (AOs) targeting these regulatory elements are listed at the bottom, including bifunctional AOs (gray ovals) and conventional AOs (open rectangles).

FIG. 2.

Diagrammatic representation of the positions of AOs targeting the ISS-N1 region. The sequence of exon 7 is shown in upper case and that of intron 7 in lower case. The sequence of ISS-N1 and the annealing sites for the 18-mer ASO-10–27, 20-mer HSMNEx7D, and 25-mer PMO25 are highlighted. Note: In the Morpholinos the uridine (U) is replaced by thymine (T).

FIG. 3.

PMO25 promotes exon 7 inclusion in vitro. (A) Cell-free in vitro splicing assay using radiolabeled SMN2 transcripts combined with PMO18, PMO20, or PMO25 at concentrations between 0 and 500 nM. (B) Graph showing ratio of SMN2 exon 7 inclusion to exclusion with increasing concentrations of PMOs. The relative ratio labeled on the y axis indicates that the readings have been normalized against the reading obtained for the no-PMO (0 nM) sample. (C) A representative reverse transcription-PCR image shows the effect of PMOs on SMN2 exon 7 inclusion in cultured SMA fibroblasts. Cells were treated at 100 and 500 nM in each PMO group. (D) Quantitative real-time PCR assay of the relative expression of full-length SMN2 transcript against the Δ7 SMN2 transcript in PMO-treated SMA fibroblasts. (E) Western blotting of SMN protein in PMO-treated SMA fibroblasts. (F) Semiquantitative analysis of SMN protein expression normalized to tubulin. *p<0.05; **p<0.01; ***p<0.001.

FIG. 4.

Effects of PMO18 and PMO25 on exon 7 inclusion and human SMN protein expression in SMA transgenic mice. (A) Newborn SMA transgenic mice that carried two copies of the human SMN2 gene were administered, by intracerebroventricular (ICV) injection, PMO18 or PMO25 at 20 μg/g on postnatal day (PND) 01. Exon 7 inclusion in brain and spinal cord was analyzed by reverse transcription-PCR 10 and 20 days postinjection. Representative RT-PCR gel images are displayed. (B) Quantitative real-time PCR analyses of exon 7 inclusion in brain and spinal cord, 10 and 20 days after a single ICV injection of saline or either PMO18 or PMO25 at 20 μg/g on PND0. *p<0.05; **p<0.01. (C) Quantitative real-time PCR analyses of exon 7 inclusion in brain and spinal cord, examined from PND10 to PND40 at 10-day intervals, after a single ICV injection of PMO25 (20 μg/g) on PND0. (D) Newborn SMA transgenic mice that carried two copies of the human SMN2 gene were given PMO18 or PMO25 at 40 μg/g by ICV injection on PND0. Quantitative real-time PCR analyses of exon 7 inclusion in brain and spinal cord were performed 20 days later. *p<0.05. (E) Western blotting of human SMN protein expressed in brain from PMO-treated SMA transgenic mice. The expressed protein was probed by a human-specific SMN antibody in samples extracted from mice treated by ICV injection of PMO25 or PMO18 (20 μg/g) on PND0. (F) Semiquantitative analysis of human SMN protein expression normalized to tubulin. *p<0.05; **p<0.01; ***p<0.001.

FIG. 5.

Survival curves and weight gain of type I SMA mice treated with PMO18 and PMO25 by ICV injection. (A) Survival curves of type I SMA mice treated with a single ICV injection of PMO18 or PMO25 at 20 μg/g on PND0. (B) Survival curves of type I SMA mice treated with a single ICV injection of PMO18 or PMO25 at 40 μg/g on PND0. (C) Body weight gain up to 30 days in type I mice treated with PMOs at 20 μg/g. Less weight gain was observed in PMO18-treated mice, corresponding to their poor survival. (D) Body weight gain up to 30 days in type I mice treated with PMOs at 40 μg/g. Saline-injected type I mice and heterozygous control mice [(SMN2)₂^+/–; smn^+/–] were used as body weight controls.

FIG. 6.

Efficacies of various delivery routes in type I SMA mice. Type I SMA mice were administered PMO25 (40 μg/g) by a single ICV injection on PND0, or by single systemic delivery (via the facial vein) on PND0, or by repeat systemic deliveries on PND0 (via the facial vein) and PND3 (subcutaneously or intraperitoneally). (A) Survival curves of type I SMA mice treated with PMO25 via different delivery routes at 40 μg/g. (B) Body weight gain in type I mice treated with PMO25 at 40 μg/g by single ICV, single intravenous, or repeated systemic injections. (C) Representative reverse transcription-PCR gel images show exon 7 inclusion in CNS tissues (brain and spinal cord) and peripheral tissues (TA muscle, heart, liver, and kidney) 10 days after the repeat systemic administration. (D) Quantitative analyses of exon 7 inclusion by real-time PCR in CNS and peripheral tissues after systemic administrations. *p<0.05; **p<0.01; ***p<0.001.

FIG. 7.

Efficacy of VMO25 in type I and mild SMA mice. (A) Survival curves of type I mice after systemic delivery of VMO25 or PMO25 on PND0. (B) Tail rescue by VMO25 in mild SMA mice. The genotype of mouse 1 was (SMN2)₂^+/+; smn^+/–, used as wild-type control; the genotype of mice 2 and 3 was (SMN2)₂^+/+; smn^–/–, that is, mild SMA mice. Single facial vein injections of saline into mice 1 and 2, and of VMO25 (10 μg/g) into mouse 3, were conducted on PND0. Images of the mice were captured at ages of 3 weeks, 4 weeks, 8 weeks, and 9 months. Tail necrosis in mouse 2 (arrowed) had clearly progressed with time. VMO25-treated mild SMA mouse 3 (asterisked) had a slightly shorter but normal-looking tail compared with wild-type mouse 1. (C) Effect of systemically delivered VMO25 in young adult mice on exon 7 inclusion in CNS and peripheral tissues. VMO25 (10 or 5 μg/g) was injected into young adult heterozygous SMA mice on PND21 via the tail vein. Brain, spinal cord, skeletal muscle (TA), heart, liver, and kidney were collected 10 days after the injection. Exon 7 inclusion in these tissues was analyzed by reverse transcription-PCR and quantified by real-time PCR. Representative reverse transcription-PCR pictures are displayed. (D) The ratio of full-length SMN2 transcript to Δ7 SMN2 transcript was quantified by real-time PCR (*p<0.05; ***p<0.001). (E) Survival curves of type I SMA mice treated with a single ICV injection of VMO25 at 4 or 2 μg/g.