Journal: RNA Biology

Article Title: Nusinersen: the antisense oligonucleotide at the forefront of spinal muscular atrophy treatment

doi: 10.1080/15476286.2026.2675858

Figure Lengend Snippet: Alternative splicing of SMN1 and SMN2 transcripts and the effects of nusinersen. (A) Genomic overview of SMN1 (upper) and SMN2 (lower) genes. Exons are shown as colored boxes, introns as broken lines. C6 ( SMN1 ) and C6U ( SMN2 ) are labeled. After transcription, exon 7 of SMN1 is fully included, generating full-length transcript, while exon 7 of SMN2 is predominantly skipped, forming the SMN2∆7 mRNA. (B) Effect of nusinsersen on splicing of SMN2 exon 7. A closeup depicting the sequence of SMN2 exon 7 and flanking intronic sequences is shown. Exon 7 is boxed and indicated with uppercase letters, while intronic sequences are shown with lowercase letters. Exonic positions are numbered relative to the 3′ss of exon 7. Upstream intronic positions are numbered with negative numbers relative to the 3′ss, while downstream intronic positions are numbered with positive numbers relative to the 5′ss. ISS-N1 is indicated in red. Nusinersen base pairs with ISS-N1 (base pairs indicated with black lines) and inhibits its negative effects, restoring predominant exon 7 inclusion.

Article Snippet: Singh and UMASS Medical School, are currently benefiting from licencing of the ISS-N1 target to Ionis Pharmaceuticals/Biogen.

Techniques: Alternative Splicing, Labeling, Sequencing

Journal: RNA Biology

Article Title: Nusinersen: the antisense oligonucleotide at the forefront of spinal muscular atrophy treatment

doi: 10.1080/15476286.2026.2675858

Figure Lengend Snippet: The impact of the last position of SMN2 exon 7 on alternative splicing. (A) Splicing of wild type SMN2 exon 7. Top panel portrays regulatory elements of SMN2 exon 7. Negative sequences (exinct, 3′ cluster, and ISS-N1) identified by in vivo selection are boxed in red, while positive sequences (conserved tract) are boxed in green. C6U and A54 residues are circled in red. Other coloring and labeling are the same as in . Lower panel: base pairing of wild type U1 snRNA (wtU1) with the wild type SMN2 exon 7 5′ss. Canonical base pairs are indicated with black dots, while G:U wobble base pairs are indicated with red circles. The a residue at the last position of exon 7 does not base pair with the cognate base in wtU1, potentially limiting recruitment. (B) Splicing of the A54G mutant of SMN2 exon 7. Top panel portrays regulatory elements of SMN2 exon 7 when A54G mutation is present. A54G is circled in green, other labels and colors are the same as (A). Lower panel: base pairing of wtU1 with the 5′ss of the A54G mutant of SMN2 . mutation provides another strong G:C base pair between wtU1 and the 5′ss, fully restoring exon 7 inclusion.

Article Snippet: Singh and UMASS Medical School, are currently benefiting from licencing of the ISS-N1 target to Ionis Pharmaceuticals/Biogen.

Techniques: Alternative Splicing, In Vivo, Selection, Labeling, Residue, Mutagenesis

Journal: RNA Biology

Article Title: Nusinersen: the antisense oligonucleotide at the forefront of spinal muscular atrophy treatment

doi: 10.1080/15476286.2026.2675858

Figure Lengend Snippet: Proposed mechanism of nusinersen action is mediated by RNA structure and RNA-binding proteins. (A) Sequence and structural context of the 5′ss of SMN2 exon 7. The last 17 bases of exon 7 are circled. Structural elements TSL2, TSL3, and ISTL1 are labeled. Canonical base pairs are indicated with black lines while G:U/G:T wobble base pairs are indicated with red circles. Negative sequence elements are boxed in red while positive elements URC1 and URC2 are boxed in green. Numbering is the same as in . hnRNPA1/A2 and potentially other protein factors interact with ISS-N1 and prevent interaction of positive splicing factors TIA1/TIAR. (B) Sequence and structural context of the SMN2 exon 7 5′ss when nusinersen is present. Nusinersen base pairs with ISS-N1and prevents formation of both TSL3 and ISTL1 while also blocking the interaction of protein factors, allowing TIA1/TIAR to bind to URC1 and URC2.

Article Snippet: Singh and UMASS Medical School, are currently benefiting from licencing of the ISS-N1 target to Ionis Pharmaceuticals/Biogen.

Techniques: RNA Binding Assay, Sequencing, Labeling, Blocking Assay

Journal: RNA Biology

Article Title: Nusinersen: the antisense oligonucleotide at the forefront of spinal muscular atrophy treatment

doi: 10.1080/15476286.2026.2675858

Figure Lengend Snippet: Engineered U1 (eU1) snRNAs targeting sequences at and near the 5′ss restore SMN2 exon 7 inclusion. Top left: interaction of wild type U1 snRNA (wtU1) with the SMN2 exon 7 5′ss. Canonical base pairs are indicated with black dots while G:U wobble base pairs are indicated with red circles. Top right: eU1 restoring full complementarity to the 5′ss of SMN2 exon 7 predominantly restores exon 7 inclusion. Lower panel: six eU1s targeting different GU dinucleotides have different impacts of SMN2 exon 7 splicing. ISS-N1 is indicated with a red box. Isoforms are shown below the base pairing diagram, ‘-’ indicates decreased isoform amount, ‘+’ indicates increased isoform amount. Two eU1s trigger usage of a cryptic 5′ss in intron 7 at position 23 (Cr1), which is indicated in the diagram with a short line in between exon 7 and exon 8 boxes.

Article Snippet: Singh and UMASS Medical School, are currently benefiting from licencing of the ISS-N1 target to Ionis Pharmaceuticals/Biogen.

Techniques:

Journal: RNA Biology

Article Title: Nusinersen: the antisense oligonucleotide at the forefront of spinal muscular atrophy treatment

doi: 10.1080/15476286.2026.2675858

Figure Lengend Snippet: ISS-N1 has been the target of a wide range of ASO chemistries and ASOs carrying terminal modifications. (A) The chemical structures of five different ASO chemistries that have all been used for SMN2 exon 7 splicing correction. (B) Graphical overview of PMO ASOs with different end modifications to facilitate transport into cells and across the BBB.

Article Snippet: Singh and UMASS Medical School, are currently benefiting from licencing of the ISS-N1 target to Ionis Pharmaceuticals/Biogen.

Techniques:

Journal: RNA Biology

Article Title: Nusinersen: the antisense oligonucleotide at the forefront of spinal muscular atrophy treatment

doi: 10.1080/15476286.2026.2675858

Figure Lengend Snippet: Overview of transcriptome-wide off-target effects of three ISS-N1-targeting ASOs. Base pairing of ASOs to ISS-N1 are shown, with the magnitude of off-target effects indicated to the right. Upregulated genes are indicated with green up arrows, while downregulated genes are indicated with red down arrows. Increased exon inclusion events are indicated with blue up arrows, while increased exon skipping is indicated with orange down arrows. (B) Mechanism of off-target effects of F18MOE/nusinersen on exon skipping. ASO binds to exonic sequences of off-target exons, masking ISS-N1-like exonic splicing enhancers (ESEs) and triggering exon skipping.

Article Snippet: Singh and UMASS Medical School, are currently benefiting from licencing of the ISS-N1 target to Ionis Pharmaceuticals/Biogen.

Techniques:

Journal: RNA Biology

Article Title: Nusinersen: the antisense oligonucleotide at the forefront of spinal muscular atrophy treatment

doi: 10.1080/15476286.2026.2675858

Figure Lengend Snippet: Shorter ASOs minimize hybridization-mediated off-target effects of nusinersen. Left: base pairing of F18MOE/nusinersen as well as shorter 14-mer and 10-mer sequences to ISS-N1 downstream of SMN2 exon 7. All three ASOs trigger increased inclusion of SMN2 exon 7. Right: base pairing of F18MOE/nusinersen as well as shorter 14-mer and 10-mer sequences to the off-target POLR2H exon 2. Due to mismatches and wobble base pairs between ASOs and the off-target exon, only the full-length 18mer has any effect on splicing.

Article Snippet: Singh and UMASS Medical School, are currently benefiting from licencing of the ISS-N1 target to Ionis Pharmaceuticals/Biogen.

Techniques: Hybridization