Clinical Applications of Oligonucleotides
Oligonucleotide-based therapeutics have made rapid progress in the clinic for the treatment of a variety of disease indications. These therapeutics either use unmodified DNA or RNA or closely related compounds. From development and regulatory perspective, they fall somewhere between small molecules and biologics. Several of these compounds are in clinical development and many have received regulatory approval for human use. With their specificity, functional diversity and limited toxicity, therapeutic oligonucleotides hold enormous promise.
Oligonucleotides to Down-Regulate Gene Expression
The earliest developed antisense oligonucleotides (ASOs) sought to modulate gene expression through inhibition of protein translation via the steric hindrance of translation initiation sites within the 5' untranslated regions of the target gene. This strategy of gene down-regulation has since been replaced by the exploitation of endogenous cellular machinery to induce RNA interference (RNAi) or RNase H based mechanisms of gene down-regulation for therapeutic purposes. RNAi effector molecules, known as small interfering RNA (siRNA), are typically 2'-mer double-stranded RNA ASOs that are recruited into the RNA-induced silencing complex (RISC) to effectively cleave the target mRNA. Despite the early promise of RNAi in pre-clinical studies, to date, only a small handful of siRNAs have had success in clinical trials.
Except for the RNAi pathway, ASOs can be exploited to induce targeted gene knockdown through the recruitment of endogenous RNase H1 to degrade mRNA at sites of DNA: RNA hybridization caused by ASO binding. Typically, these ASOs are modified to have a chimeric design of a DNA core to induce RNase H activity, flanked by 2' modified 'wings' such as 2'-O-methoxyethyl, to improve drug stability and tissue distribution. Mipomersen is the second antisense drug approved by the FDA and is a 2'-mer 'gapmer' complementary to the coding region of apoB mRNA for use as an adjunct therapy in familial hypercholesterolemia.
Figure 1. Gene regulation mechanisms mediated by antisense oligonucleotides (ASOs).
Oligonucleotides Targeting MicroRNA
The role of microRNAs (miRNAs), short endogenous non-coding RNAs, in the regulation of many disease processes present them as an attractive target for therapeutic modulation. miRNAs exert their action by guiding a miRNA-induced silencing complex to 3' UTRs of target mRNAs to induce mRNA decay and/or translational repression of the target gene. One of the most promising approaches to modulate miRNA expression is the development of single-stranded ASOs that directly bind target miRNAs to inhibit their function (anti-miRs), and thus derepress their target gene. Similar to other ASO-based approaches, anti-miRs have to be chemically modified to enhance their pharmacokinetic properties, binding affinity and resistance to nucleases in an in vivo environment. One of the earliest successesin vivo was the use of cholesterol-conjugated 2'-O-Me phosphorothioate modified antagomirs targeting miR-122, amongst others, that demonstrated antagomir-specific reduction across a wide range of tissues.
Oligonucleotides Modulate Splicing
Mutations that disrupt natural pre-mRNA splicing are the underlying genetic cause for a number of genetic diseases. One approach to address this is the development of ASOs to modulate pre-mRNA splicing in the target gene to by-pass the disease-causing mutation. The most well-studied example to date is for Duchenne muscular dystrophy (DMD), whereby frame-shift deletion or non-sense mutations in the DMD gene result in a loss of functional dystrophin protein, leading to a fatal progressive muscle wasting disease. A milder allelic form of the disease, Becker muscular dystrophy (BMD), also exists whereby in-frame mutations allow the production of an internally deleted, yet partially functional protein with generally slower disease progression. By applying ASOs to induce removal of exons around a frame-shift deletion or containing a nonsense mutation ('exon skipping'), it is possible to restore the mature mRNA reading frame to produce an internally deleted protein similar to that observed in BMD. Numerous proof-of-concept studies have been demonstrated in murine and canine models of DMD.
Reference
- McClorey, G.; et al. (2015). An overview of the clinical application of antisense oligonucleotides for RNA-targeting therapies. Current opinion in pharmacology. 24: 52-58.
