Mechanistic principles of antisense targets for the treatment of spinal muscular atrophy

Abstract

Spinal muscular atrophy (SMA) is a major neurodegenerative disorder of children and infants. SMA is primarily caused by low levels of SMN protein owing to deletions or mutations of the SMN1 gene. SMN2, a nearly identical copy of SMN1, fails to compensate for the loss of the production of the functional SMN protein due to predominant skipping of exon 7. Several compounds, including antisense oligonucleotides (ASOs) that elevate SMN protein from SMN2 hold the promise for treatment. An ASO-based drug currently under Phase III clinical trial employs intronic splicing silencer N1 (ISS-N1) as its target. Cumulative studies on ISS-N1 reveal a wealth of information with significance to the overall therapeutic development for SMA. Here, the authors summarize the mechanistic principles behind various antisense targets currently available for SMA therapy.

Figure 1. Targets of SMA therapy

(A) Relative position of major targets for an ASO-mediated splicing correction in SMA. Neutral and positive numbering start from the first positions of exon 7 and intron 7, respectively. Negative numbering starts from the last position of intron 6. Element 1 occupies positions from −68 to −112 in intron 6 [57]. ISS-N1 and GCRS occupy positions from +10 to +24 [58] and from +7 to +14 [59] in intron 7, respectively. 5′ISTL1 and 3′ISTL1 refer to the 5′ and 3′ strands of ISTL1, respectively [60]. An ASO-based approach has been used to target the 100^th position of intron 7 [61]. A+100G refers to an SMN2 specific mutation at the 100^th position of intron 7 [62]. TSL2 is an inhibitory stem-loop structure that sequesters the 5′ ss of exon 7 [63]. (B) Relative location of cis-elements in the context of the RNA secondary structure of exon 7 and intron 7. Numbering is the same as in panel A. Presented structures of exon 7 and intron 7 were deduced from enzymatic and chemical structure probing [60,63].

Figure 2. Effect of ISS-N1 and GCRS-targeting ASOs on accessibility of the 5′ ss of exon

The SMN2 mRNA is numbered from the start of exon 7 (neutral numbers) and the start of intron 7 (positive numbers) with a rainbow scheme ranging from blue to red in the 5′ to 3′ direction for the cartoon depiction of the phosphate backbone in the 3D models. Nucleotide bases in all figures are colored by type: green for adenine, blue for cytosine, orange for guanine and red for uracil. Secondary structures include base pairing used for model prediction indicated by a solid dash and additional base pairs formed during model calculations denoted by a tilde. All model calculations included a construct of the 5′ ss of exon 7 comprising 100 nucleotides ranging from 33^rd position in exon 7 to +300^th position in intron 7 with a 225 nucleotide deletion between +57^th and +283^rd replaced by a three nucleotides to complete a UAAC tetraloop. A hierarchical stepwise assembly protocol within the Rosetta modeling suit (version 2015.09.57646) was used to build local secondary structure motifs and assemble the motifs into a tertiary model that was further refined by energy minimization [92]. (A) In the absence of ASO binding, the 3D model of the 5′ ss of exon 7 (shown on the right) was calculated with experimental base pairing restraints derived from SHAPE-based chemical probing of the secondary structure (shown on the left). The model prediction includes two stem-loop hairpin motifs, TSL2 (38^th position in exon 7 to +2^nd position in intron 7) and TSL3 (+17^th to +41^st positions); and two long-range stem interactions, ISTL1 (+3^rd to +10^th and +290^th to +297^th positions) and ISTL2 (+50^th to +56^th and +283^rd to +289^th positions). (B) Predicted changes in the three dimensional structure of the 5′ ss of exon 7 upon binding of an ISS-N1 targeting 18-mer ASO (F18) (shown on the right) include disruption of the TSL3 motif and ISTL1 structure. Annealing position of F18 is marked on the secondary structure (shown left). (C) The three dimensional structure of the 5′ ss of exon 7 upon binding of an ISS-N1 targeting 8-mer ASO (F8) (shown right) predicts retention of all stem-loop motifs and long-range stem interactions. Annealing position of F8 is marked on the secondary structure (shown left). (D) Predicted changes in the three dimensional structure of the 5′ ss of exon 7 upon binding of a GCRS targeting 8-mer ASO (3UP8) (shown right) include disruption of the ISTL1 structure. Annealing position of 3UP8 is marked on the secondary structure (shown left).

Figure 3. Mechanism of SMN2 exon 7 splicing correction by an ISS-N1-targeting ASO

Proposed mechanism by which an ISS-N1-targeting ASO promotes exon 7 inclusion. An ISS-N1 targeting ASO sequesters binding sites of hnRNP A1/A2, disrupts/destabilizes TSL3, ISTL1 and ISTL2. This leads to binding of TIA1 to now accessible sites (shown in green) and enhanced recruitment of U1 snRNP to the 5′ ss of exon 7. Mechanism is adapted from [60]. Numbering is the same as in Figure 1A.

Figure 4. Effect of a dual-masking ASO on accessibility of the 5′ ss of exon 7

Annealing of a dual-masking ASO (Dual-1) to ISS-N1 and the 3′ ss of exon 8 brings the 5′ ss of exon 7 and 3′ ss of exon 8 in close proximity. Dual-1 is described in [61]. Remaining structure of intron 7 is shown. Numbering is the same as in Figure 1A.