Using antisense oligonucleotides in combination or in higher concentration to treat spinal muscular atrophy (SMA) may actually be detrimental, according to a new study published in Genes.

The results show that antisense oligonucleotides used in combination or at high concentration seem to reduce the rate of splicing and lead to the formation of cryptic exons or splicing variants that may introduce frameshifts or stop codons in the mRNA.

Antisense oligonucleotides have been used for some time to treat SMA. However, they are usually not efficient in patients who are in the more advanced stages of the disease. The current regimen of treatment can also sometimes lead to a ceiling effect, so researchers have hypothesized that higher doses or combinations of antisense oligonucleotides may increase their effectiveness.

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Here, a team of researchers led by Masakazu Shinohara, MD, PhD, from the Department of Community Medicine and Social Healthcare Science at Kobe University Graduate School of Medicine in Hyogo, Japan used SMA fibroblasts to test this hypothesis.

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The researchers used a double combination of 3 different antisense oligonucleotides at 3 different concentrations. One of these, like nusinersen, an approved SMA treatment, targets intronic splicing suppressor site N1 in intron 7 while the other 2 targets the 3′ splice site and 5′ region of exon 8.

The results showed that a lower concentration of antisense oligonucleotides (50 nM or 100 nM) led to better splicing than a higher concentration (200 nM) of treatment. This was the case for all 3 antisense oligonucleotides tested.

Moreover, each antisense oligonucleotide resulted in a cryptic exon in exon 6 at a high concentration. Adding a mixture of 2 different antisense oligonucleotides, even at the intermediate dose of 100 nM each, led to the inclusion of the cryptic exon in the mRNA.

“Although it is difficult to directly apply the results of cultured cell studies to the clinical setting, our data suggested that a high concentration of [antisense oligonucleotides] or their use in combination may show unanticipated effects,” the researchers concluded.


Wijaya YOS, Niba ETE, Nishio H, et al. High concentration or combined treatment of antisense oligonucleotides for spinal muscular atrophy perturbed SMN2 splicing in patient fibroblasts. Genes. 2022;13(4):685. doi:10.3390/genes13040685