Splice-switching antisense oligonucleotides as therapeutic drugs

Abstract

Splice-switching oligonucleotides (SSOs) are short, synthetic, antisense, modified nucleic acids that base-pair with a pre-mRNA and disrupt the normal splicing repertoire of the transcript by blocking the RNA-RNA base-pairing or protein-RNA binding interactions that occur between components of the splicing machinery and the pre-mRNA. Splicing of pre-mRNA is required for the proper expression of the vast majority of protein-coding genes, and thus, targeting the process offers a means to manipulate protein production from a gene. Splicing modulation is particularly valuable in cases of disease caused by mutations that lead to disruption of normal splicing or when interfering with the normal splicing process of a gene transcript may be therapeutic. SSOs offer an effective and specific way to target and alter splicing in a therapeutic manner. Here, we discuss the different approaches used to target and alter pre-mRNA splicing with SSOs. We detail the modifications to the nucleic acids that make them promising therapeutics and discuss the challenges to creating effective SSO drugs. We highlight the development of SSOs designed to treat Duchenne muscular dystrophy and spinal muscular atrophy, which are currently being tested in clinical trials.

Figure 1.

Splice-switching oligonucleotides (SSOs) modulate alternative splicing. (top) Diagram of a pre-mRNA transcript with exons depicted as gray boxes and introns as lines. An intronic splicing silencer (ISS, red) and exonic splicing enhancer (ESE, green) are shown bound by a trans-acting inhibitory splicing factor protein (red oval) or stimulatory splicing factor (green oval). These SF proteins either block (−) or promote (+) splicing at splice sites bordering the surrounding exons. (left panel) An SSO that base-pairs to a splicing enhancer sequence creates a steric block to the binding of the stimulatory splicing factor to its cognate enhancer binding site. This block thereby disrupts splicing and results in exon skipping. (right panel) In contrast, an SSO that base-pairs to a splicing silencer sequence element blocks splicing silencer activity by preventing binding of a negatively acting splicing factor. Disruption of the binding of splicing inhibitory proteins to its cognate binding sequence activates splicing at the splice site that is negatively regulated by the silencer element, resulting in exon inclusion.

Figure 2.

Structures of oligonucleotide analogs commonly used in splice switching applications in vivo. Modifications that are used in the SSOs presented in Table 1 are depicted. Unmodified RNA is shown for reference. Base refers to unmodified adenine, cytosine, guanine or uracil.

Figure 3.

Splice-switching antisense oligonucleotides (SSOs) mechanism of action. SSOs can gain entry into cells in vivo following injection of a naked/unformulated ASO into the blood or cerebrospinal fluid. SSOs can be bound by circulating proteins and have been proposed to enter into cells by binding to receptors for these proteins on the cell surface. Subsequently, SSOs undergo compartmentalization followed by vesicle release at which point they are free to move into the nucleus, bind pre-mRNA and induce a splicing switch that results in an mRNA that is translated into a protein isoform in the cytoplasm.

Figure 4.

Schematic representation of disease associated splicing in DMD (top panel) and SMA (bottom panel) and the SSO targeting strategy used to therapeutically switch splicing for the treatment of the disease. Boxes are exons and horizontal lines are introns. Splicing regulatory sequences and protein regulators are noted. ESE, exonic splicing enhancer; ISS, intronic splicing silencer N1. SMNΔ7 refers to a form of SMN lacking amino acids encoded by exon 7. DystrophinΔ refers to a form of dystrophin truncated after amino acids encoded by exon 52 before encountering a premature termination codon in exon 52. Dystrophin isoform refers to a form of the Dystrophin protein encoded by mRNA lacking exons 50 and 51. The position of the stop codon is indicated by a red hexagon.