SMA is the second most common genetic disorder of children and infants caused by insufficient levels of SMN protein due to the loss of the SMN1 gene. Presence of a defective gene, SMN2, makes SMA a unique genetic disease that could be avoided and possibly cured by redirecting SMN2 exon 7 splicing. Among several approaches to correct aberrant splicing, an ASO-based approach provides a superior alternative due to the anticipated target specificity. Size of an ASO is an important determinant in success of an ASO-based strategy. Despite the expected advantages, it is not known if very short ASOs could anneal to the target and bring desired changes in a sequence-specific manner, particularly at the low nanomolar concentrations.
Here we report an 8-mer ASO (3UP8) as the shortest ASO to correct the aberrant splicing of SMN2
exon 7 in SMA patient cells. To the best of our knowledge, this is the first report in which an 8-mer ASO is able to effectively correct aberrant splicing in a patient cell line. Identification of this ASO was achieved through a systematic approach of ultra-refined antisense microwalk in an intronic region adjacent to the 5' ss of exon 7. The 8-mer ASO exerts its stimulatory effect through binding to a GC-rich sequence (CUGCCAGC) spanning from the 7th
position of intron 7 (). Underscoring an evolutionary significance, this intronic region is not conserved between human and mice.42
CUGCCAGC target sequence seems to be highly accessible since low nanomolar concentrations of 3UP8 fully restores SMN2
exon 7 inclusion (). Consistently, the predicted secondary structure puts this target sequence in an internal loop flanked by terminal stem-loop structures ().28
Our ultra-refined antisense microwalk with about 50 ASOs captured relative strength of multiple antisense targets that differed by a single nucleotide. As a consequence, it also revealed positions of high significance, wherein sequestering of the last five residues (CCAGC) of the GC-rich target was found to be absolutely required for the stimulatory response on SMN2
exon 7 inclusion (). Hence CCAGC residues could be considered as the core motif, analogous to the seed sequence of the micro-RNA target.50
However, unlike microRNAs that require assembly of a RNA-induced silencing complex (RISCs) on an 18-mer or longer sequence, our antisense response is solely based on the short RNA:RNA duplex. Based on the published reports, it is highly unlikely that protein factors could form a stable complex with a short RNA:RNA duplex. However, we cannot rule out the possibility of secondary contacts that might have been affected.
The GC-rich target described here does not resemble any known binding motif of a splicing factor, although, it overlaps with the first five residues of ISS-N1, an intronic element that harbors two putative hnRNP A1 binding sites.42,43
The C residue at the first position (1
C) of ISS-N1 is not the part of hnRNP A1 motif, yet sequestering of this position was found to be absolutely necessary for the antisense response. Further, several ASOs that did not sequester 1
C produced an inhibitory effect even though they fully sequestered both hnRNP A1 motifs (data not shown). These results suggest that the stimulatory response of ASOs is a combination of effects not necessarily caused by blocking of hnRNP A1 motifs.
Various mechanisms may account for the stimulatory response exerted by 3UP8. The most obvious among them is the strong target affinity of 3UP8 compared to an inhibitory factor that may transiently interact with the same target during the dynamic process of splicing. It is also possible that the RNA:RNA duplex formed between 3UP8 and the GC-rich target helps bring a subtle change in the RNA structure in the vicinity of the 5' ss. Such a structural change may help improve U1 snRNP recruitment and/or the 5' ss recognition. We have previously shown that recruitment of U1 snRNP at the 5' ss of exon 7 is a limiting step for SMN2
exon 7 inclusion.13,29
Our results also suggest that the catalytic core of splicing is not affected by a RNA:RNA duplex formed between an ASO and its target immediately downstream of the U1 snRNA binding site. However, dissociation of ASO from the target sequence through a helicase reaction during the catalytic core formation could not be ruled out. In this scenario, the same antisense will be recycled several times on different SMN2
pre-mRNAs. This is an obvious advantage of short ASOs in an ASO-based therapy because frequency of drug (ASO) administration could be minimized.
Our work underscores the high target specificity of very short ASOs during RNA:RNA interactions. For instance, a single mismatch in the middle of the target caused a drastic decrease in the stimulatory response by 3UP8. On the contrary, longer ASOs tolerated this mismatch mutation due to a large duplex formed between an ASO and the target. Tolerance of mismatched mutations provides an inherent drawback and therapeutic risk associated with longer ASOs. Consistently, high concentrations of a 20-mer ASO (Anti-N1) targeting intron 7 produced an off-target effect on SMN2 exon 3 splicing, whereas identical concentrations of 3UP8 had no effect ().
Owing to the high target specificity and an efficient antisense response by a short ASO, 3UP8 increased levels of SMN in SMA patient cells. It also restored levels of several key proteins that are generally downregulated in SMA (). These include factors involved in snRNP biogenesis (Gemin 2 and Gemin 8) and RNA splicing (Tra2-β1 and hnRNP Q).38,55
hnRNP Q proteins have been also implicated in other aspect of RNA metabolism, such as RNA transcription, translation, stability and trafficking.53-55
Increase in ZPR1 in 3UP8-treated cells suggests that a short ASO is capable of restoring SMN-interacting factors, reduced expressions of which are associated with the progressive loss of motor neurons.56,57
Despite a gradual decrease in the levels of SMN2
exon 7 inclusion after two days, high SMN levels were maintained for five days after single treatment with 40 nM 3UP8. These findings suggest a substantially longer half-life of SMN owing to the stabilization of SMN through association with itself and/or with other factors. Consistent with the restoration of the SMN-interacting partners, 3UP8-treated cells showed increased numbers of sub-nuclear bodies (gems) in the nucleus ().
Currently SMA has no cure, although several small compounds capable of increasing levels of SMN in SMA have been identified.23
Mechanisms of actions and side effects of these compounds remain unknown. Earlier ASO-based strategies promised high target specificity and focused on large ASOs in the anticipation that small motifs could not be targeted by small ASOs.42-49
In general, literature is replete with studies using 15-mer or longer ASOs for modulation of alternative splicing. Our work provides the first evidence of high target specificity for a very short ASO and sets a unique precedence of pathogenic splicing modulation by RNA molecules less than half the size of the most reported ASOs. Compared to large ASOs that carry the inherent risk of partial sequestration of different kinds of small motifs and tolerate mismatch mutations, we show that the stimulatory activity of a small ASO is exclusively dependent upon the perfect match with a single motif that is uniquely located within an accessible region of a negative context. Short ASOs offer additional advantages including low cost of synthesis, ease of chemical modifications, reduced chances of immune response, and higher probability of crossing biological barriers.58
When promotion of exon inclusion is the goal, a short intronic target brings the desired benefits of noninterference with nuclear export and translation. Hence, our findings represent further advancement towards an ASO-based therapy of SMA and bring a unique perspective to our understanding of splicing regulation of a defective gene associated with a major genetic disease of children and infants.