This is the first demonstration of a technique designed to enhance trans
-splicing efficiency by blocking downstream splice site selection. While the initial trans
-splicing RNA alone showed considerable activity in cell-based models of SMA in terms of significantly increasing SMN levels and increasing snRNP activity 
, these results did not translate to the in vivo
context. Our research highlights a key finding that low dose trans
-splicing is enhanced by the co-expression of the ASO and resulted in detectable levels of SMN expression in vivo
As a therapeutic approach, trans
-splicing offers the advantage over gene replacement in that expression is intrinsically controlled by the endogenous promoter 
. Consequently, temporal and spatial constraints on gene expression are retained. As SMN
expression has previously been shown to be significantly down-regulated from embryogenesis to adulthood, this additional restraint may prove beneficial for long-term exposure to a SMN trans
-splicing vector. Avenues of research directed to the reduction of tsRNA vector dosage would consequently inhibit the negative effects of off-target trans
-splicing over greater amounts of time.
The genetic context of SMA represents an intriguing target for a number of therapeutic strategies, including trans-splicing. In the SMN2 gene, the intrinsic quality of the exon 7 splice acceptor site is reduced due to the C/T transition. Therefore, the competition between SMN2 cis-splicing and trans-splicing is likely reduced, providing a potential advantage to trans-splicing in the SMN context compared to other alternatively regulated exons that retain fully functional splice sites. We propose trans-splicing is further enhanced relative to cis-splicing with the introduction of an inhibitory ASO. Since the tsRNA annealing domain hybridizes over the target exon, it is logical that this may promote exon 7 skipping. Therefore, the addition of the anti-sense likely blocks this escape pathway and further promotes trans-splicing. Unlike many ASOs that modify splicing, the ASOs used in this context are native RNA structures, not oligonucleotides with modified backbones. Therefore, the affects may be difficult to observe at steady-state levels when used as an ASO alone. However, given that this sequence overlaps the intron 7/exon 8 boundary, it was not surprising that the ASO recapitulated the increase in trans-splicing observed with the slow polymerase and intron 7-deleted construct.
In general, the field of trans
-splicing therapeutics can benefit from the discovery of combined ASO-tiling and tsRNAs. ASO-tiling increases the potential of tsRNA by modulating cis
-splicing signals within the target transcript. Additionally, in a novel application of ASO-In411
co-delivery targeting a upstream SMN
intron 3 exon 4 splicing context produced enhancement of basal trans
-splicing. (Supplemental Figure S2
) Studies here demonstrate the limitations of dual vector delivery in difficult fibroblast transfection conditions. Co-transfection dynamics hinder the potential of ASO/tsRNA pre-mRNA redirection and can be circumvented by sub-cloning of the single vector pMU3. The combination of ASO-tsRNA mechanism and a novel single vector platform produces a potent enhancement of basal trans
-splicing. In the case of trans
-splicing RNAs designs such as, 5′ or 3′ versions would require examination of beneficial upstream and downstream sequences to tile with ASO RNA.
The in vivo results demonstrate ASO-tsRNA represents a tractable therapeutic yet highlight the transient nature of plasmid transfections. The use of AAV vectors for the delivery of gene therapies would provide a substantial longevity to ASO-tsRNA expression and thus SMN2 redirection. Future studies could examine the role of expression of the virus vectors and the SMA CNS. We conclude the combined effects of ASO-tsRNAs in a novel expression vector enhanced trans-splicing in the context of SMN2 alternative cis-splicing. Applications of this biotechnology on a well characterized SMA model mouse demonstrate the promise of in vivo restoration of SMN via trans-splicing RNAs.