DG9-conjugated morpholino rescues phenotype in SMA mice by reaching the CNS via a subcutaneous administration
Tejal Aslesh, Esra Erkut, Jun Ren, Kenji Rowel Q. Lim, Stanley Woo, Susan Hatlevig, Hong M. Moulton, Simon Gosgnach, John Greer, Rika Maruyama, Toshifumi Yokota
Tejal Aslesh, Esra Erkut, Jun Ren, Kenji Rowel Q. Lim, Stanley Woo, Susan Hatlevig, Hong M. Moulton, Simon Gosgnach, John Greer, Rika Maruyama, Toshifumi Yokota
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Research Article Genetics Muscle biology

DG9-conjugated morpholino rescues phenotype in SMA mice by reaching the CNS via a subcutaneous administration

Abstract

Antisense oligonucleotide–mediated (AO-mediated) therapy is a promising strategy to treat several neurological diseases, including spinal muscular atrophy (SMA). However, limited delivery to the CNS with AOs administered intravenously or subcutaneously is a major challenge. Here, we demonstrate a single subcutaneous administration of cell-penetrating peptide DG9 conjugated to an AO called phosphorodiamidate morpholino oligomer (PMO) reached the CNS and significantly prolonged the median survival compared with unconjugated PMO and R6G-PMO in a severe SMA mouse model. Treated mice exhibited substantially higher expression of full-length survival of motor neuron 2 in both the CNS and systemic tissues compared with nontreated and unmodified AO–treated mice. The treatment ameliorated the atrophic musculature and improved breathing function accompanied by improved muscle strength and innervation at the neuromuscular junction with no signs of apparent toxicity. We also demonstrated DG9-conjugated PMO localized in nuclei in the spinal cord and brain after subcutaneous injections. Our data identify DG9 peptide conjugation as a powerful way to improve the efficacy of AO-mediated splice modulation. Finally, DG9-PMO is a promising therapeutic option to treat SMA and other neurological diseases, overcoming the necessity for intrathecal injections and treating body-wide tissues without apparent toxicity.

Authors

Tejal Aslesh, Esra Erkut, Jun Ren, Kenji Rowel Q. Lim, Stanley Woo, Susan Hatlevig, Hong M. Moulton, Simon Gosgnach, John Greer, Rika Maruyama, Toshifumi Yokota

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Figure 1

Subcutaneous administration of DG9-PMO at PD0 extends survival and improves motor function in severe SMA mice.

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Subcutaneous administration of DG9-PMO at PD0 extends survival and impro...
(A) Survival curves of heterozygous mice (Smn+/– SMN2Tg/–) (Hets), nontreated (NT) mice, unconjugated PMO (PMO), DG9-PMO, and MOE injected at PD0 at a dose of either 40 or 80 mg/kg. For 40 mg/kg studies, n = 15 (Hets), n = 22 (NT), n = 49 (unconjugated PMO), n = 14 (DG9-PMO), n = 29 (MOE). For 80 mg/kg studies, n = 15 (Hets), n = 22 (NT), n = 7 (unconjugated PMO), n = 6 (DG9-PMO), n = 4 (MOE) (*P < 0.05, **P < 0.01, ****P ≤ 0.0001, log-rank Mantel-Cox test). (B) Weight of mice at PD7 administered with either 40 or 80 mg/kg doses. Each dot (symbol) indicates a neonatal pup. (C) Hind limb suspension assay (HLS). Mice were treated with 40 mg/kg AOs at PD0. Score is based on the position of the hind limbs when suspended from a tube (n = 12–20 mice per group). (D) Righting reflex test. Mice were treated with 40 mg/kg AOs at PD0. The ability of mice to right themselves on their paws was measured every alternate day PD2 to PD20 (left) (n = 12–20 mice per group). The mean righting reflex time at PD6 and PD8 was also indicated (right: box-and-whiskers plots). Box edges, 25th and 75th percentiles; central line, median; whiskers, range. (E) Forelimb grip strength measured in adult mice at PD30 and PD60 from 40 mg/kg treatment groups normalized to the body weight. In B, D (box-and-whisker plots), and E, 1-way ANOVA followed by post hoc Tukey’s test was performed. Single symbols represent P < 0.05, double symbols represent P < 0.01, and triple symbols represent P < 0.005. In C and D (left graph), 2-way ANOVA followed by Holm-Šídák multiple comparison was performed. Single symbols represent P < 0.03; double symbols represent P < 0.002; triple symbols represent P < 0.0002. *NT, #PMO, @DG9-PMO, &MOE. Error bars: SEM.