Multiple strategies aimed at correcting exon 7 splicing of SMN2,
a modifying gene for SMA, have been investigated. Mechanistic studies to understand how SMN2
exon 7 is alternatively spliced led to the discovery of several cis
-elements and trans
-acting factors that can be targeted to stimulate exon 7 inclusion [42
]. A number of drugs, including synthetic compounds that can modify SMN2
splicing, have been identified using cell-based high-throughput drug screening [42
]. On the basis of the current knowledge of splicing mechanisms, we and others have employed antisense-based technologies to stimulate exon 7 inclusion. In recent years, a growing number of studies have demonstrated that MOE-modified ASOs (with a phosphodiester or a phosphorothioate backbone) and antisense PNAs can be valuable tools, not only for dissecting gene function, but also for clinical applications [19
]. Our previous work showed that ESSENCE compounds have the potential to treat diseases caused by exon skipping resulting from the loss of ESEs in mutant genes, including SMN2
]. A critical parameter in ESSENCE effectiveness is the selection of the optimal binding site along a target exon. In principle, the best target sequences are those that comprise negative exonic splicing signals or secondary structures, such as ESSs.
In this study, we systematically analyzed SMN2 exon 7 with a large number of MOE ASOs to identify putative ESSs. We employed a two-step ASO walk method, with an initial coarse ASO walk in 5-nt steps along the entire exon, followed by high-resolution single-nucleotide walks within the regions identified in the first step. With the first-step walk, we identified two ASO targets of potential therapeutic importance. The second-step microwalks optimized the ASOs and defined the apparent boundaries of the two ESS-containing regions. Using three independent splicing assays, we identified two potent ASOs that have significant therapeutic potential for SMA treatment. Our data also suggest that the two-step ASO walk is a powerful general method that can be used for screening inhibitory or stimulatory splicing regions present in the target exon(s) and the surrounding intron sequences of any gene.
Our data revealed an essential core sequence in the center of the 54-nt exon 7 from +22 to +33 (GAAGGAAGGTGC), which is surrounded by two separate inhibitory regions (A and B) containing negative splicing signals, with region A extending close to the upstream 3′ splice site (+4 to +21) and region B extending close to the downstream 5′ splice site (+34 to +51) (). Blocking any part of the central core sequence promoted efficient skipping of exon 7, whereas blocking either of the two inhibitory regions promoted exon 7 inclusion. The 5′ portion of the central core sequence comprises a previously identified Tra2β1-binding motif (GAAGGA) [38
]; the 3′ part (AGGTGC) may represent another cis
-acting element that is also crucial for exon 7 recognition. A relatively long conserved sequence in the middle of exon 7, surrounded by two short inhibitory sequences, was previously identified by an iterative in vitro selection method [43
]. Through an analysis of mutability, three segmental sequences with negative or positive cis
-acting elements were inferred: the most conserved residues are located in the middle and form a conserved tract (+16 to +44), whereas the highly mutable upstream nucleotides form an inhibitory region (+3 to +15), and seven highly mutable downstream nucleotides (+45 to +51) form another negative element [43
]. Our data sharpen the boundaries for these three segments, as verified by three different splicing assays and several ASOs, particularly the two most effective ASOs, 07–21 and 34–48. The target sequence of the 15-mer ASO 07–21 overlaps by six nucleotides with the previously reported conserved tract, and 11 out of 15 target nucleotides of ASO 34–48 are part of this conserved track. Nucleotides A36 and C37, which fall within the inhibitory region B, have been suggested to be part of a stimulatory motif, as the double mutation A36U/C37U abolishes exon 7 inclusion in the SMN1
]. However, the double mutation might create a stronger inhibitory motif, or strengthen a secondary RNA structure that impairs exon 7 recognition.
Schematic Diagram of the In Vivo Effects of All Tested ASOs on Exon 7 Inclusion
We searched exon 7 for putative ESSs with the Web servers PESX (http://cubweb.biology.columbia.edu/pesx
) and ACESCAN2 (http://genes.mit.edu/acescan2/index.html
). PESX found no ESS motifs, whereas ACESCAN2 found three putative ESS motifs: GGTTTT (+1 to +6), TTTTAG (+3 to +8) and TTCCTT (+39 to +44). The first two motifs partially overlap with the inhibitory region A, and the last motif resides within the inhibitory region B, so these motifs might contribute to the inhibitory properties of regions A and B.
We note that both optimized ASOs are 15-mers, whereas all five of the 18-mer ASOs (36–53, 35–52, 34–51, 33–50, and 32–49) displayed more or less inhibitory effects on SMN2 exon 7 inclusion. The longer 18-mer ASOs are likely to overlap two or more binding sites, resulting in complex effects. The strong inhibitory effect of ASO 32–49 and the relatively weak inhibitory effect of ASO 33–50 might be explained by their interference with the positive regulation conferred by the essential core sequence, because the 5′-most boundary nucleotide of their target sequence is 33G and 34C, respectively. The other three ASOs, whose target sequences lie next to the exon–intron junction, might interfere with recognition of the 5′ splice site.
A small fraction of the ASOs had inconsistent effects on exon 7 splicing in the different splicing assays. One type of inconsistency occurred between the minigene splicing assays (both in vitro and in vivo) and the endogenous gene assay. The other type of inconsistency occurred between the in vitro splicing assay and the in vivo splicing assays. The first type of inconsistency may reflect the differences in the pre-mRNA substrates, although SMN2
exon 7 was identical in all the assays. In both the in vitro splicing assay and the in vivo minigene splicing assay, a small minigene was used. Higher-order structure differences between the small minigene pre-mRNA and the much larger endogenous pre-mRNA might affect the accessibility of some ASOs to their target sequences in exon 7, resulting in splicing differences. The second type of inconsistency might reflect differences in the splicing-reaction environments between the cell-free splicing assay and the cell-based splicing assays, even though in general, the in vitro splicing reactions accurately reproduce cellular splicing events. For example, it is possible that the effects of some ASOs are more sensitive to changes in the concentration of certain splicing factors resulting from the nuclear extraction procedure. Another possibility is that the discrepancies result from kinetic differences and the coupling between transcription and mRNA processing that normally occurs in vivo, but not in standard in vitro splicing reactions [44
]; moreover, transcription from different promoters—as in the minigene and the endogenous gene—can also affect use of alternative cassette exons in vivo [45
Why ASOs 07–21 and 34–48 are more effective than the original ones, which bind to their target sequences only one or two nucleotides upstream or downstream, is currently under investigation. Because the ASOs have the same lengths and the same or similar G/C contents, annealing kinetics is probably not the cause, assuming equal accessibility to their target sites in exon 7. Two possible explanations of the effects of ASOs 07–21 and 34–48 are that they precisely block one or more splicing silencers, or efficiently disrupt an inhibitory RNA secondary structure. It has been reported that hnRNP A1 can inhibit SMN2
exon 7 inclusion [12
]. In region A of SMN2,
SF2/ASF presumably fails to block the cooperative propagation of hnRNP A1, due to the loss of an SF2/ASF motif caused by the C6T transition [13
]. ASO 07–21 binds a 15-nt sequence that spans the mutant SF2/ASF motif; therefore, it is reasonable to assume that one of the roles played by ASO 07–21 is similar to a proposed function of SF2/ASF in SMN1
exon 7, i.e., blocking the propagation of hnRNP A1. ASO 07–21 might also block other inhibitory motifs residing in region A, for which an extended inhibitory context has been proposed [46
]. We have unexpectedly observed in microwalk A that the 12-mer ASO 04–15 inhibited exon 7 inclusion; ASO 09–20 was likewise inhibitory, at least in vivo. These results suggest that the inhibitory effect of region A is the result of interplay among multiple trans
-acting factors. It is likely that ASOs 04–15 and 09–20 can effectively block an undefined weak stimulatory signal, but cannot efficiently block the propagation of hnRNP A1 and possibly other repressors. A terminal-stem loop has been predicted in the less-studied region B, extending to two intron 7 nucleotides [47
]. Though it is possible that the inhibitory property of region B is attributable to the proposed RNA secondary structure itself, other possibilities cannot be ruled out, including the existence of one or more splicing repressors that recognize a specific motif or RNA structure. Additional experimental studies will be required to elucidate the precise splicing-stimulatory mechanism of ASO 34–48.
We cannot exclude the possibility that the stimulatory ASOs act in part by stabilizing the pre-mRNAs and/or mRNAs to which they hybridize. Because only some of the exon 7 ASOs stimulate exon 7 inclusion, such a stabilization effect would presumably involve masking of specific instability elements. However, the effect of the ASOs is primarily, if not exclusively, at the level of splicing, because an effect at the level of mRNA half-life should result in the same proportional increase in SMN1 and SMN2 full-length mRNAs, yet the effects on SMN1 were much smaller, consistent with a switch in alternative splicing from the low basal level of exon 7–skipped mRNA. In addition, we did not observe any ASO-mediated stabilization of the labeled pre-mRNA in the in vitro splicing experiments.
We used three different assays to demonstrate that the ASOs that promote exon inclusion are compatible with synthesis of full-length SMN protein, indicating that they do not block mRNA export or translation. First, we showed that ASOs 07–21 and 34–48 promote efficient expression of reporter proteins corresponding to exon 7 inclusion in HEK293 cells. Second, we showed that the same ASOs promote expression of full-length SMN protein in primary fibroblasts from a type I SMA patient; though significant, this effect appears subtle because of the relatively low transfection efficiency with these cells. Finally, we showed that the same ASOs promote an increase in the number of nuclear gems detected with anti-SMN antibody in the patient fibroblasts; this single-cell immunofluorescence assay obviates the problem of transfection efficiency and provides an indirect readout for SMN protein levels [4
Our initial goal in this study was to define optimal antisense targets to maximize ESSENCE effectiveness. However, our optimized antisense targets should also be useful with bifunctional ASOs [30
]. The antisense portions of the bifunctional ASOs used in two earlier studies were not optimized. The first study used a 15-mer ASO targeting the exon 7 sequence +2 to +16 [30
], i.e., the same target sequence as that of our ASO 02–16. However, we found that this ASO had a slightly negative effect on exon 7 inclusion in vivo with both the SMN2
minigene and the endogenous SMN2
gene, though it had a slightly stimulatory effect on splicing in vitro (B, A, and 5B). ASO 02–16 was one of few ASOs that gave inconsistent effects among the different splicing assays we used. The second study used a 20-mer ASO targeting the exon 7 sequence +6 to +25 [31
], which partially overlaps the central core sequence that is essential for exon 7 inclusion. On the basis of our results, we predict that more-effective bifunctional ASOs targeting exon 7 can be designed, using the ASO 07–21 or 34–48 sequences as the antisense moiety.
Although we expect that even more-potent effectors can be designed by attaching ASOs 07–21 or 34–48 to activation-domain peptides or SR protein–binding sites—as in the ESSENCE [34
] and TOES [29
] methods—these ASOs appear to be sufficiently effective on their own to be tested in animal models of SMA. ASOs with the same chemistry were recently demonstrated to be active modulators of splicing in mice [48
]. More generally, the antisense walk strategy described here should be applicable to identifying exon-targeting ASOs that modulate alternative splicing of normal or mutant genes. At the same time, these ASOs can facilitate the identification of important cis
-regulatory sequences that can be further investigated to elucidate splicing mechanisms, and to identify trans
-acting factors that may be useful molecular targets for disease therapy.