Systemic peptide-mediated oligonucleotide therapy improves long-term survival in spinal muscular atrophy

Abstract

The development of antisense oligonucleotide therapy is an important advance in the identification of corrective therapy for neuromuscular diseases, such as spinal muscular atrophy (SMA). Because of difficulties of delivering single-stranded oligonucleotides to the CNS, current approaches have been restricted to using invasive intrathecal single-stranded oligonucleotide delivery. Here, we report an advanced peptide-oligonucleotide, Pip6a-morpholino phosphorodiamidate oligomer (PMO), which demonstrates potent efficacy in both the CNS and peripheral tissues in severe SMA mice following systemic administration. SMA results from reduced levels of the ubiquitously expressed survival motor neuron (SMN) protein because of loss-of-function mutations in the SMN1 gene. Therapeutic splice-switching oligonucleotides (SSOs) modulate exon 7 splicing of the nearly identical SMN2 gene to generate functional SMN protein. Pip6a-PMO yields SMN expression at high efficiency in peripheral and CNS tissues, resulting in profound phenotypic correction at doses an order-of-magnitude lower than required by standard naked SSOs. Survival is dramatically extended from 12 d to a mean of 456 d, with improvement in neuromuscular junction morphology, down-regulation of transcripts related to programmed cell death in the spinal cord, and normalization of circulating insulin-like growth factor 1. The potent systemic efficacy of Pip6a-PMO, targeting both peripheral as well as CNS tissues, demonstrates the high clinical potential of peptide-PMO therapy for SMA.spinal muscular atrophy | survival motor neuron | antisense oligonucleotide | splice switching oligonucleotide | cell-penetrating peptide S pinal muscular atrophy (SMA), a leading genetic cause of infant mortality primarily due to lower motor neuron degeneration and progressive muscle weakness, results from loss of the ubiquitous survival motor neuron 1 gene (SMN1) (1, 2). Humans have a second nearly identical copy, SMN2, that differs from SMN1 by a crucial nucleotide transition within exon 7 leading to the predominant generation of an alternative exon 7-excluded transcript and only marginally functional protein (3-7). SMN2 therefore fails to compensate for loss of SMN1 unless sufficient copies are present to generate functional levels of full-length SMN protein (2).A rational, gene therapy-based approach for SMA uses singlestranded antisense splice-switching oligonucleotides (SSOs) to enhance SMN2 pre-mRNA exon 7 inclusion via steric block of splice regulatory pre-mRNA elements (8). Targeting the intron splice silencer N1 (ISS-N1) site within intron 7, by deletion or SSOmediated splice switching, improves exon 7 inclusion (9, 10). ISS-N1-targeted SSOs used to treat presymptomatic severely affected neonatal SMA mice, via systemic or intracerebroventricular administration, extend survival from 10 to >100 d (11, 12). Although SSO targeting to the CNS is essential, there is also evidence for a peripheral role for the SMN in SMA (13-24).Although SSO therapy is currently at an advanced stage of de...

Efficient SMN Rescue following Subcutaneous Tricyclo-DNA Antisense Oligonucleotide Treatment

“…Recently, a peptide-conjugated PMO AON (Pip6a-PMO) has been administered intravenously in SMA mice at P0 and P2, achieving significant lifespan extension 46 . More importantly, this group demonstrated corrected SMN2 transcripts in the CNS after tail vein administration at 7.5 weeks, suggesting that this AON may penetrate the BBB.…”

ISS-N1 makes the first FDA-approved drug for spinal muscular atrophy

“…The exclusive licensing of ISS-N1-targeting ASOs from UMass Medical School allows IONIS Pharmaceuticals to develop additional drugs based on ISS-N1 target. Studies in the laboratories of Dr. Arthur Burghes at The Ohio State University and Dr. Francesco Muntoni at University College London independently validate the efficacy of ISS-N1-targeting morpholino ASOs [53–59]. SMA patients will tremendously benefit if additional antisense drugs based on morpholino and other chemistries are developed.…”

“…ASOs targeting ISS-N1 are predicted to enhance SMN2 exon 7 inclusion by at least two mechanisms; first, by blocking binding of hnRNP A1 to two target motifs in the region [35], second, by causing secondary structural rearrangements and preventing an inhibitory long-distance interaction with downstream sequences deep within intron 7 [37, 38, 39]. Numerous studies have demonstrated the efficacy of ASOs targeting ISS-N1 in both SMA patient cells and mouse models of SMA and using multiple ASO chemistries [28, 34, 35, 44, 49–59). Based on the number of the independent studies performed, ISS-N1 would easily rank as the most studied antisense target for splicing correction for human disease.…”

How the discovery of ISS-N1 led to the first medical therapy for spinal muscular atrophy

“…For instance, Wood and colleagues have recently reported promising CNS delivery of systemically-administered peptide-conjugated PMOs. 70,71 However, the pharmacokinetics and tolerability of these conjugated ASOs remains to be further examined in non-human primates. Nevertheless, these modifications could be particularly informative for other genetic diseases in which nucleic-acid-based treatment may be beneficial.…”