Identification of a Peptide for Systemic Brain Delivery of a Morpholino Oligonucleotide in Mouse Models of Spinal Muscular Atrophy

Abstract

Splice-switching antisense oligonucleotides are emerging treatments for neuromuscular diseases, with several splice-switching oligonucleotides (SSOs) currently undergoing clinical trials such as for Duchenne muscular dystrophy (DMD) and spinal muscular atrophy (SMA). However, the development of systemically delivered antisense therapeutics has been hampered by poor tissue penetration and cellular uptake, including crossing of the blood-brain barrier (BBB) to reach targets in the central nervous system (CNS). For SMA application, we have investigated the ability of various BBB-crossing peptides for CNS delivery of a splice-switching phosphorodiamidate morpholino oligonucleotide (PMO) targeting survival motor neuron 2 (SMN2) exon 7 inclusion. We identified a branched derivative of the well-known ApoE (141-150) peptide, which as a PMO conjugate was capable of exon inclusion in the CNS following systemic administration, leading to an increase in the level of full-length SMN2 transcript. Treatment of newborn SMA mice with this peptide-PMO (P-PMO) conjugate resulted in a significant increase in the average lifespan and gains in weight, muscle strength, and righting reflexes. Systemic treatment of adult SMA mice with this newly identified P-PMO also resulted in small but significant increases in the levels of SMN2 pre-messenger RNA (mRNA) exon inclusion in the CNS and peripheral tissues. This work provides proof of principle for the ability to select new peptide paradigms to enhance CNS delivery and activity of a PMO SSO through use of a peptide-based delivery platform for the treatment of SMA potentially extending to other neuromuscular and neurodegenerative diseases.

Keywords: brain delivery; peptides; spinal muscular atrophy; splice-switching oligonucleotide; survival motor neuron.

FIG. 1.

Splice-switching activity of P-PMO conjugates in H2K mdx muscle cells measured using nested real time-polymerase chain reaction (RT-PCR) at 1 and 5 μM. (A) DMD exon-23 skipping of receptor-targeting peptides conjugated to PMO. (B) Exon-23 skipping activity of P-PMOs with peptides of unknown BBB crossing mechanism. (C) Branched and tandem dimers of ApoE conjugates (0.5 and 1 μM) showing higher exon-23 skipping activities than single ApoE-PMO (1 and 5 μM). The branched analogue of PepC7 and the branched chimera of ApoE-PepC7-PMO showed similar exon-skipping levels to the monomer of PepC7 at 1 and 5 μM. (D) Assessment of proteolytic stability of tandem ApoE-PMO and branched ApoE-PMO (K→A) at 1 μM was measured by preincubating the P-PMOs in 50% mouse serum for 1 and 2 h. The level of DMD exon-23 skipping was significantly decreased after 1 h and remained unchanged after 2 h. *P < 0.05, **P < 0.001, ***P < 0.0001 cf. PMO (1 μM); ^#P < 0.05, ^##P < 0.001 cf. PMO (5 μM). BBB, blood–brain barrier; DMD, Duchenne muscular dystrophy; PMO, phosphorodiamidate morpholino oligonucleotide; P-PMO, peptide-PMO.

FIG. 2.

Effect of P-PMO on the viability of human hepatocyte (Huh7) cells. All P-PMOs were tested at a relatively high concentration of 40 μM and the cell viability was determined using an MTS assay. P-PMO conjugates showed no sign of cytotoxicity apart from branched and tandem ApoE, which caused significant cell death with <40% cell viability. However, replacement of K → A in branched ApoE led to enhanced cell viability of >85%. MTS, 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium.

FIG. 3.

Effect of P-PMO conjugate treatment on SMN2 exon 7 inclusion in cultured SMA patient-derived fibroblast cells. The average percentage increase in full-length SMN2 at three different concentrations of P-PMO conjugates was determined by densitometric analysis of RT-PCR gel images. SMA, spinal muscular atrophy; SMN2, survival motor neuron 2.

FIG. 4.

(A) In vivo screening of P-PMO conjugates targeting SMN2 exon 7 inclusion in SMA mice at PND4. The increase of full-length SMN2 transcript in the brain, kidney, and quadriceps following facial vein injection was determined using real-time reverse transcriptase quantitative polymerase chain reaction (RT-qPCR). The branched ApoE (K→A)-PMO was the only P-PMO, which showed a significant increase in the levels of full-length SMN2 transcript in all three tissues compared with the control (saline and naked PMO)-treated groups. (B) Level of full-length SMN2 transcript in newborn SMA mice treated with P-PMO measured using RT-qPCR. The change in the level of full-length SMN2 transcripts expression was normalized to the total SMN2 transcripts. Data are presented as mean ± SEM. *P < 0.05, **P < 0.001 cf. saline and naked PMO-treated groups. PND, postnatal day; SEM, standard error of the mean.

FIG. 5.

Treatment of severe SMA (Smn1^{tm1Hung/tm1Hung};SMN2^tg/o) mice with two doses of 10 mg/kg at PND0 and PND2 with Br-ApoE (K→A)-PMO administered through facial vein injection resulted in a marked increase in (A) the median survival to 78 days, and (B) steady increased weight gain compared with the untreated, naked PMO-treated, and Br-ApoE (K→A)-scrambled-PMO-treated groups. (C) Muscle strength, measured by the hindlimb suspension “tube test,” was maintained for Br-ApoE (K→A)-PMO until PND12, whereas the saline, naked, and Br-ApoE (K→A)-scrambled-PMO showed a gradual decline in muscle strength. (D) The Br-ApoE (K→A)-PMO-treated pups showed improvement in their righting ability.

FIG. 6.

Quantitative real-time PCR analysis of relative expression of full-length SMN2 in the brain, spinal cord, and kidney of adult SMA mice treated with Br-ApoE (K→A)-PMO with 6 weekly doses of 10 mg/kg and harvested 7 days post final administration. Data are presented as mean ± SEM. ^#P < 0.01, *P < 0.001, **P < 0.0001 cf. saline-treated groups. PCR, polymerase chain reaction.