LNA/DNA mixmer-based antisense oligonucleotides correct alternative splicing of the SMN2 gene and restore SMN protein expression in type 1 SMA fibroblasts

Abstract

Spinal muscular atrophy (SMA) is an autosomal recessive disorder affecting motor neurons, and is currently the most frequent genetic cause of infant mortality. SMA is caused by a loss-of-function mutation in the survival motor neuron 1 (SMN1) gene. SMN2 is an SMN1 paralogue, but cannot compensate for the loss of SMN1 since exon 7 in SMN2 mRNA is excluded (spliced out) due to a single C-to-T nucleotide transition in the exon 7. One of the most promising strategies to treat SMA is antisense oligonucleotide (AON)-mediated therapy. AONs are utilized to block intronic splicing silencer number 1 (ISS-N1) on intron 7 of SMN2, which causes exon 7 inclusion of the mRNA and the recovery of the expression of functional SMN protein from the endogenous SMN2 gene. We developed novel locked nucleic acid (LNA)-based antisense oligonucleotides (LNA/DNA mixmers), which efficiently induce exon 7 inclusion in SMN2 and restore the SMN protein production in SMA patient fibroblasts. The mixmers are highly specific to the targeted sequence, and showed significantly higher efficacy than an all-LNA oligonucleotide with the equivalent sequence. These data suggest that use of LNA/DNA mixmer-based AONs may be an attractive therapeutic strategy to treat SMA.

Estimated number of off‐target candidate sites for antisense oligonucleotides in human mRNA sequences

“…Furthermore, Shimo et al. () and Touznik, Maruyama, Hosoki, Echigoya, and Yokota () showed that 13‐mer SSOs with LNA modifications efficiently induced exon skipping and exon inclusion at the cellular level, respectively. These 13‐ to 16‐mer ASOs would theoretically have several tens to hundreds of complementary regions if one or more mismatches are tolerated (Table ).…”

“…For example, IONIS-STAT3-2.5 RX /AZD9150 and ISTH0036 are gapmer ASOs under clinical development and are a 16-mer and 14-mer, respectively. Furthermore, Shimo et al (2014) and Touznik, Maruyama, Hosoki, Echigoya, and Yokota (2017) showed that 13-mer SSOs with LNA modifications efficiently induced exon skipping and exon inclusion at the cellular level, respectively. These 13-to 16-mer ASOs would theoretically have several tens to hundreds of complementary…”

Multiple competing RNA structures dynamically control alternative splicing in human ATE1 gene

“…The cloned fragment of Ate1 lacks a substantial part of intron 7, which may affect splicing. We therefore independently examined the role of R1, R3, and R4 in the endogenous Ate1 transcript using locked nucleic acid (LNA)/DNA mixmers as antisense oligonucleotides (AONs) that interfere with RNA secondary structure (38). Since R1 and R4 overlap with splice sites, we chose to use AON with specific sequence complementary to R3 (AON1) ( Figure 2G).…”

Mutations in spliceosome genes and therapeutic opportunities in myeloid malignancies

“…Another avenue to therapeutically target splicing activity in cancers is the selective targeting of the pathological splicing events with oligonucleotide‐based approaches. The prototype for this class of therapies is the antisense oligonucleotides (ASOs) that have recently been approved in the United States for the treatment of Duchenne muscular dystrophy and spinal muscle atrophy (SMA) . These oligonucleotides hybridize to RNA and can either target it for degradation or be used to affect splicing of pre‐mRNA.…”

“…The prototype for this class of therapies is the antisense oligonucleotides (ASOs) that have recently been approved in the United States for the treatment of Duchenne muscular dystrophy 139 and spinal muscle atrophy (SMA). 140 These oligonucleotides hybridize to RNA and can either target it for degradation or be used to affect splicing of pre-mRNA. In the case of SMA, the approved oligonucleotide targets an intronic splicing silencer of SMN2 without degrading the pre-mRNA and allows for inclusion of an exon that results in increased stability of the protein.…”