Several antisense oligonucleotide drugs using this type of cleavage have been approved by the US FDA including mipomersen and inotersen. Mipomersen is the second antisense oligonucleotide drug approved by the FDA (2013) as an adjunct to lipid-lowering therapy for the treatment of homozygous familial hypercholesterolemia (HoFH) (Figure 3). Apolipoprotein B-100 (ApoB-100) is the main component of low-density lipoprotein and its precursor very low-density lipoprotein (VLDL). Mipomersen binds to ApoB-100 mRNA and cleaves its sequence, thereby reducing lipid levels in these individuals (Clarke et al.,2023).

The second group of antisense oligonucleotide drugs primarily regulates the splicing of pre-mRNAs by utilizing a steric hindrance-based mechanism. These ASOs bind to specific sequences within the pre-mRNA transcripts, and subsequently, modulate the other splicing factors to produce alternative splicing (Figure 3). A few FDA-approved ASOs which work on this mechanism include nusinersen, eteplirsen, golodirsen, and casimersen (Kim, 2022).

Individuals with spinal muscular atrophy have variations in SMN1, which encodes the survival motor neuron (SMN) protein. These variations prevent the expression of a functional SMN protein from the SMN1 gene locus but some amount of SMN protein is still produced from the SMN2 locus. However, this protein product is smaller and less stable because of exon 7 skipping in the SMN2 pre-mRNA. As a result, there is loss of motor neurons eventually leading to muscle wasting. The intron splicing enhancer located between exons 7 and 8 in SMN2 pre-mRNA leads to this exon 7 skipping during the splicing of SMN2 pre-mRNA. Nusinersen is an antisense oligonucleotide that binds to this element and blocks its recognition by the splicing factors. As a result, the SMN2 pre-mRNA is spliced like SMN1 pre-mRNA leading to the production of a more stable SMN protein. This drug has shown improved motor function in infants with spinal muscular atrophy in a phase III clinical trial (Clarke et al.,2023) and was approved by the US FDA for clinical use in 2016.

Exon skipping approach has been utilized by a series of antisense oligonucleotide-based drugs such as eteplirsen, golodirsen, and casimersen that have been developed for the treatment of Duchenne muscular dystrophy (DMD) (Zhang et al.,2023). In individuals with DMD, the mRNA coding for dystrophin protein usually harbours a variation that can alter the reading frame, thereby producing a truncated (non-functional) protein in these individuals. Eteplirsen binds to the exonic splicing enhancer present in exon 51 of DMD pre-mRNA. Exon splicing enhancer element is required for the inclusion of this exon in the mature mRNA. Thus, binding of eteplirsen to this element leads to exon 51 skipping and correcting the reading frame in the mature mRNA. As a result, there is production of a short but functional dystrophin protein in these individuals. Exon 51 skipping may be beneficial for individuals with DMD with exon deletions ending at exon 50 or starting at exon 52 (Łoboda et al.,2020). Golodirsen and casimersen also utilize a similar mechanism in a different subset of individuals. Golodirsen induces exon 53 skipping while casimersen leads to exon 45 skipping to produce a functional version of the dystrophin protein (Kim, 2022).

Several clinical trials using antisense oligonucleotides such as IONIS-HTTRx for Hungtington disease and Tofersen, which targets the SOD1 protein frequently implicated in familial amyotrophic lateral sclerosis are underway (Clarke et al.,2023).

Small interfering RNAs (siRNA) Small interfering RNAs (siRNAs) and micro RNAs (miRNAs) are small duplex RNA molecules that target messenger RNA (mRNA) leading to post-transcriptional gene silencing. siRNAs and miRNAs utilize the RNA interference (RNAi) pathway to modulate the expression of their target mRNA. RNAi is the endogenous and intrinsic defence mechanism of the body against invading viruses and transposable elements (Chen et al.,2018). In the RNAi pathway, endogenous small RNAs (siRNA or miRNA) form a complex with Argonaute 2 protein (Ago2) to produce an RNA-induced silencing complex (RISC) (Figure 4). This complex binds to the target mRNA via sequence-specific binding leading to mRNA cleavage (Zhang et al.,2023).

siRNAs are double-stranded RNA molecules about 18 to 25 nucleotides in length. Four siRNA-based drugs have been approved by the US FDA namely patisiran, givosiran, inclisiran, and lumasiran for the treatment of hereditary transthyretin-mediated (hATTR) amyloidosis, acute hepatic porphyria, heterozygous familial hypercholesterolemia (HeFH) and primary hyperoxaluria type 1, respectively (Kim, 2022). However, no miRNA-based drug has been by approved so far by the US FDA for the treatment of monogenic disorders.

Patisiran is the first FDA-approved siRNA-based drug (2018) for treating hereditary transthyretin-mediated (hATTR) amyloidosis. This condition is caused by variation in the transthyretin (TTR) gene leading to the production of a misfolded transthyretin protein and it eventually leads to amyloid deposition in various tissues of the body. Patisiran utilizes a lipid nanoparticle-based delivery system and is injected into the body via intravenous infusion. These particles enter the hepatocytes via ApoE (apolipoprotein E) receptors. In the hepatocytes, patisiran combines with RISC and this complex binds to the 3’ untranslated regions (UTR) of both the wild-type as well as mutant TTR mRNA leading to the suppression of TTR protein translation and an overall reduction in the amyloid deposition in the tissues (Zhang et al.,2023).

Aptamers Aptamers are small, single-stranded oligonucleotides (DNA or RNA) that bind to their targets (proteins, peptides, or nucleic acids) with high specificity and affinity and modulate their functions (Figure 5). Pegaptanib is the only aptamer drug that has been approved by the US FDA. It is a 28-nucleotide construct with two polyethylene glycol moieties (PEG) attached at its end. It binds to the vascular endothelial growth factor (VEGF) thereby inhibiting the interaction of VEGF with its receptor leading to the suppression of downstream VEGF signalling and cell proliferation. Pegaptanib was developed for the treatment of neovascular age-related macular degeneration, but it is rarely used nowadays because of the availability of several antibody-based drugs with similar efficacy. Nevertheless, it is a promising therapeutic strategy, and several RNA-based aptamers are under development for the treatment of various monogenic disorders (Zhu et al.,2022; Kim, 2022).

6 Conclusion

After several decades of development, RNA-based therapeutics are now becoming a clinical reality. The field of RNA-based therapy is undergoing a major expansion and the underlying potential of these therapies for personalized medicine will certainly ensure the continued development of RNA-based therapeutics for years to come.

References