Redirecting splicing with bifunctional oligonucleotides

Abstract

Ectopic modulators of alternative splicing are important tools to study the function of splice variants and for correcting mis-splicing events that cause human diseases. Such modulators can be bifunctional oligonucleotides made of an antisense portion that determines target specificity, and a non-hybridizing tail that recruits proteins or RNA/protein complexes that affect splice site selection (TOSS and TOES, respectively, for targeted oligonucleotide silencer of splicing and targeted oligonucleotide enhancer of splicing). The use of TOSS and TOES has been restricted to a handful of targets. To generalize the applicability and demonstrate the robustness of TOSS, we have tested this approach on more than 50 alternative splicing events. Moreover, we have developed an algorithm that can design active TOSS with a success rate of 80%. To produce bifunctional oligonucleotides capable of stimulating splicing, we built on the observation that binding sites for TDP-43 can stimulate splicing and improve U1 snRNP binding when inserted downstream from 5′ splice sites. A TOES designed to recruit TDP-43 improved exon 7 inclusion in SMN2. Overall, our study shows that bifunctional oligonucleotides can redirect splicing on a variety of genes, justifying their inclusion in the molecular arsenal that aims to alter the production of splice variants.

The emerging role of alternative splicing in senescence and aging

“…Although senescence and aging affect a wide‐range of biological processes, interventions that prevent or encourage the production of specific splice variants may help counteract the emergence of age‐related phenotypes and disorders. Splice‐switching antisense oligonucleotides (SSOs) that block or stimulate the use of a splice site are now used with success in animal models of human diseases and are being tested in clinical trials (Brosseau et al ., ; Havens & Hastings, ). In principle, they could be used to prevent the production of pro‐senescence variants in p53, ING1, and other transcripts.…”

Targeting Splicing in the Treatment of Human Disease

“…This approach was used to redirect the splicing to favor the inclusion of endogenous exon 7 SMN2 transcript to increase the level of functional SMN protein [38] and to alter the AS of the BCL2L1 pre-mRNA to promote apoptosis in cancer cells in culture [39]. These bifunctional oligonucleotides to alter splicing decisions can be used against a wide range of targets [40]. Frequently, ASOs are chemically modified to improve binding affinity and avoid degradation.…”

Can the HIV-1 splicing machinery be targeted for drug discovery?

“…Another useful way of blocking ss is through the use of bifunctional oligonucleotides. These molecules consist of two separate oligonucleotides: one portion is the antisense of the target, which leads to the molecule being a specific blocker, and a second molecule that does not bind the target mRNA but recruits proteins or RNA–protein complexes that alter ss selection 44. The second strategy is viable because the blocking of a single SR protein can be compensated for by other SR protein in the host cell without a significant loss of function while HIV requires specific SR proteins.…”