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Review
. 2021 Feb 3;29(2):540-554.
doi: 10.1016/j.ymthe.2020.12.022. Epub 2020 Dec 25.

Clinical Applications of Single-Stranded Oligonucleotides: Current Landscape of Approved and In-Development Therapeutics

Juergen Scharner  1 , Isabel Aznarez  2
Affiliations
Review

Clinical Applications of Single-Stranded Oligonucleotides: Current Landscape of Approved and In-Development Therapeutics

Juergen Scharner et al. Mol Ther. .

Abstract

Single-stranded oligonucleotides have been explored as a therapeutic modality for more than 20 years. Only during the last 5 years have single-stranded oligonucleotides become a modality of choice in the fields of precision medicine and targeted therapeutics. Recently, there have been a number of development efforts involving this modality that have led to treatments for genetic diseases that were once untreatable. This review highlights key applications of single-stranded oligonucleotides that function in a sequence-dependent manner when applied to modulate precursor (pre-)mRNA splicing, gene expression, and immune pathways. These applications have been used to address diseases that range from neurological to muscular to metabolic, as well as to develop vaccines. The wide range of applications denotes the versatility of single-stranded oligonucleotides as a robust therapeutic platform. The focus of this review is centered on approved single-stranded oligonucleotide therapies and the evolution of oligonucleotide therapeutics into novel applications currently in clinical development.

Keywords: CpG oligonucleotides; RNase H; antisense oligonucleotides; gapmer; splicing; steric blocking.

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Conflict of interest statement

I.A. and J.S. are employees and hold shares of Stoke Therapeutics.

Figures

None
Graphical abstract
Figure 1
Figure 1
Single-Stranded Oligonucleotide Chemistries Chemical structures of ribose and backbone modifications used in single-stranded oligonucleotides compared to DNA and RNA structures are shown. Modifications highlighted in green are used in approved oligonucleotide drugs. DNA, deoxyribonucleic acid; RNA ribonucleic acid; Me, methyl; MOE, methoxyethyl; LNA, locked nucleic acid; cEt, constrained ethyl; BNA, bridged nucleic acid; PMO, phosphorodiamidate morpholino oligomer; PO, phosphodiester; PS phosphorothioate.
Figure 2
Figure 2
Mechanism of Action of Gapmer ASOs (A) Expression of an example target gene from transcription through translation of a wild-type or toxic protein. (B) Gapmer ASO with the typical structure of modified chemistry in the wings to protect the ends from nucleases and an internal stretch of DNA that leads to the formation of the RNA-DNA hybrid when bound to the target transcript. In this example, the gapmer ASO binds to an exon in the pre-mRNA and mRNA and recruits RNase H that recognizes the RNA/DNA hybrid and cleaves the RNA. The cleavage triggers RNA degradation, leading to reduction of wild-type or toxic protein levels. Gapmer ASOs can be designed to target other transcript regions, e.g., introns. Even though RNase H is more abundant in the nucleus, RNase H-mediated cleavage of RNA-DNA hybrids can also occur in the cytoplasm.
Figure 3
Figure 3
Mechanism of Action of Nusinersen for the Treatment of Spinal Muscular Atrophy (A) Region of SMN2 pre-mRNA containing exons 6, 7, and 8. SMN2 exon 7 carries a silent single nucleotide change with respect to SMN1 that causes exon 7 skipping, which leads to an unstable SMN protein. Only 10% of SMN2 pre-mRNA is properly spliced, resulting in an insufficient level of functional SMN protein to compensate for the loss of the SMN1 gene. (B) Nusinersen binding to intron 7 of the SMN2 pre-mRNA and promoting exon 7 inclusion, which leads to increased levels of SMN protein and improved motor neuron function.
Figure 4
Figure 4
Mechanism of Action of Eteplirsen for the Treatment of Duchenne Muscular Dystrophy (DMD) (A) Effect of a deletion of exons 49–50 region in the DMD gene that causes a frameshift and leads to the introduction of a premature termination codon in exon 51. The mutant mRNA is degraded in the cytoplasm by nonsense-mediated mRNA decay (NMD), and no dystrophin protein is produced, causing DMD. (B) Eteplirsen binding to exon 51, which prevents its inclusion and restores the frame. The resulting mRNA lacking exons 49–51 is translated to generate an internally truncated dystrophin protein that retains partial function.
Figure 5
Figure 5
CpG-Containing Oligonucleotides Mediated Immune Cell Stimulation Internalized CpG-containing ISOs are mainly recognized by TLR9, which activates a complex signaling cascade resulting in the nuclear translocation of transcription factors including AP1, IRF7, and nuclear factor κB (NF-κB). Transcriptional activation of pro-inflammatory genes regulates the maturation of pDCs, the activation and proliferation of B cells, as well as the production of type I interferons and Th1-type cytokines. CpG-mediated immune cell activation, as well as the subsequent secretion of cytokines, stimulates the immune response to the co-administered antigen (not shown), and it produces a more rapid and longer lasting antibody response when compared to alternative vaccine adjuvants. In addition to vaccine adjuvants, single-stranded CpG-containing ISOs are currently also being tested in cancer patients and to modulate the immune response in inflammatory diseases (Table 1).
Figure 6
Figure 6
Single-Stranded Oligonucleotide Therapies Summary of approved drugs to date and their target tissue. Milasen is a clinical investigational treatment under an Expanded Access-Investigational New Drug application.
Figure 7
Figure 7
Mechanism of Action of QR-421a, a New Application of Steric-Blocking ASOs The figure depicts a portion of USH2A pre-mRNA containing exons 12, 13, and 14 (middle) in which exon 13 carries missense (blue sign) or premature termination codon (PTC)-introducing (nonsense or frameshift, red sign) retinitis pigmentosa (RP) mutations. Exon 13 encodes half of laminin epidermal growth factor (EGF)-like domain 4, domains 5, 6, 7, and half of laminin EGF-like domain 8 (light blue rectangles highlighted in red). Normally, missense or PTC-introducing RP mutations lead to non-functional full-length or non-functional truncated usherin, respectively (top). QR-421a (+ASO) promotes skipping of exon 13 (depicted by lines connecting exons 12 and 14), leading to the generation of an usherin protein that lacks half of laminin EGF-like domain 4, as well as domains 5, 6, 7, and half of laminin EGF-like domain 8 (bottom), but maintains proper function. Green rectangle, signal peptide; purple rectangle, laminin G-like domain; orange rectangle, laminin N-terminal domain; light blue rectangles, laminin EGF-like domains; green rectangles, fibronectin type III repeats; yellow circles, laminin G; red rectangle, transmembrane domain; blue circle, PDZ binding motif.
Figure 8
Figure 8
Mechanism of Action of STK-001, a New Application of Steric-Blocking ASOs (A) Region of SCN1A wild-type pre-mRNA containing non-productive (non-coding) exon X (yellow rectangle) and flanking coding exons (brown rectangles). SCN1A pre-mRNA is alternatively spliced such that it generates a non-productive mRNA containing the non-productive exon X, which leads to the introduction of a PTC, and a productive mRNA lacking exon X. Upon export to the cytoplasm, the non-productive mRNA is degraded by nonsense-mediated mRNA decay and the productive mRNA is translated into wild-type Nav1.1 protein. The pre-mRNA carrying DS mutations undergoes the same alternative splicing processing, but the mutant productive mRNA does not produce a functional protein (not shown in the figure), leading to haploinsufficiency of Nav1.1. (B) STK-001 (ASO) binding to the non-productive exon X of the SCN1A wild-type and mutant (not shown) pre-mRNA and promotes exon X skipping, which leads to a reduction in non-productive mRNA and increased levels of productive mRNA and wild-type Nav1.1 protein to near normal levels. STK-001 leverages the wild-type gene copy to compensate for the loss-of-function mutant alleles in DS patients.
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