Oligonucleotide therapy is a new precision medicine strategy in which short nucleotides are used to precisely block genes. These synthetic oligonucleotides are less than 20 nucleotides long and they can control gene expression by linking to Watson-Crick bases in DNA, mRNA, or pre-mRNA. Oligonucleotide drugs can suppress aberrant genes and prevent disease-causing proteins from being able to replicate, offering treatment for genetic diseases, cancers and other diseases.
We can subdivide oligonucleotide therapies into the main mechanisms that are:
Antisense oligonucleotides (ASOs)
ASOs stop protein synthesis by attaching to the complementary sequence of the target mRNA and, resulting in a double-stranded molecule, preventing the mRNA from being translated or processed. It is a mechanism routinely applied to disease due to a single gene mutation. ASOs can tame gene expression by targeting pre-mRNAs or mRNAs in three distinct ways:
1) RNase H activation: ASO recruits RNase H, which cleaves the RNA strand of RNA-DNA double-stranded bodies, effectively degrading the target RNA and reducing protein translation.
2) Translation Blocking: By spatially blocking ribosome binding sites on mRNA, ASO prevents protein synthesis without causing RNA degradation.
3) Selective splicing regulation: ASOs can affect exon inclusion or exclusion, restoring or disrupting the expression of functional proteins as needed for therapeutic intervention.
Figure 1. Mechanism of antisense oligonucleotides. ASOs bind to pre-mRNA/mRNA and act in four ways to inhibit protein synthesis (A) Inhibition of 5′ capping and polyadenylation of the tail, (B) Modulation of RNA splicing, (C) Translational repression, and (D) RNase H induced degradation[1].
Small interfering RNAs (siRNAs)
siRNAs act through the mechanism of RNA interference (RNAi), where they are able to target and degrade specific mRNAs to reduce the expression of target proteins. It's common for siRNAs to go into the cell's nucleus and triple-strand together with the double-stranded genomic DNA in order to halt protein translation and transcription. This is very specific and targets disease genes without damaging off-target transcripts.
Figure 2. Mechanism of siRNA by assembly and activation of RNA- RISC[1].
miRNAs silence genes by targeting the RNA-induced silencing complex (RISC). They can shut down the activity of target genes, thereby snuffing out disease.
Figure 3. RNA interference. For simplicity, we have shown how miRNAs can mediate RNA interference in mammalian cells by causing the degradation of protein-coding transcripts[2].
Aptamers block the action of a target protein by tethering themselves directly to the protein in its three-dimensional structure. Aptamers can mimic antibodies, but they're more specific and less immunoreactive.
Figure 4. Mechanism of aptamers showing sequence folding into 3D structure formation and binding to the target[1].
Splicing regulatory oligonucleotides (SSOs)
SSOs control gene expression by manipulating the way that precursor mRNAs splice to exon or exclude exon.
Gapmers
Gapmers are a hybrid oligonucleotide, a center DNA sequence and two chemically enhanced RNA sequences. This structure combines stability with robust RNA destruction via RNase H activation for an effective way to silence disease-causing genes.
Gene Editing Oligonucleotides
Oligonucleotides, such as guide RNA (gRNA) in the CRISPR-Cas9 system, facilitate precise genome editing by directing nuclease to specific DNA sequences. It is used to repair mutations or turn off disease-causing genes. The CRISPR-Cas system allows for genome editing by instructing the Cas enzyme to attack the desired DNA fragment. Using the CRISPR-Cas system to edit the genome has become remarkably accurate and effective – thanks to the creation and use of a number of new tools. For instance, the Single Base Editor (Base Editor) and Prime Editor (Prime Editor) have allowed genome editing to be done more efficiently without introducing DSBs (double-stranded DNA breaks), reducing off-target effects. Furthermore, the CRISPR-Cas system has facilitated the assessment of mutations at scale through the use of sequencing instruments that better allow us to see the effects of genetic variation.
Oligonucleotide therapies have a number of key clinical benefits, such as superior targeting power, disease targets that are not available for small molecule therapies, and scalability in design and synthesis. But there are also several problems for oligonucleotide therapies - rapid in vivo degradability, and immune resistance - that are being addressed by chemical modifications and improved drug delivery mechanisms.
| Therapeutic Class | Disease Area | Representative Examples |
| Neurological Disorders | Spinal muscular atrophy (SMA) | ASOs like nusinersen enhance SMN2 gene expression. |
| Genetic Diseases | Transthyretin amyloidosis | siRNAs like patisiran reduce TTR protein levels. |
| Oncology | Chronic myeloid leukemia | Aptamers targeting tyrosine kinase pathways. |
| Ophthalmology | Age-related macular degeneration | VEGF-inhibiting aptamers like pegaptanib. |
| Viral Infections | Hepatitis B virus (HBV) | ASOs targeting viral replication intermediates. |
Oligonucleotide therapy must also be made safe and effective and in accordance with patients' rights and interests. For instance, since oligonucleotides can incite immune reaction or targeted toxicity, such side effects must be well-studied in clinical trials. Safety: Oligonucleotide therapies come with a set of potential hazards. These risks can include, but are not limited to:
A. While oligonucleotides can be made to go directly to target mRNA targets to minimise off-target toxicity, some risks still exist. For instance, some oligonucleotides can cross-react to sequences outside the target group, leading to gene expression adjustments that weren't intended.
B. Newly constructed molecules such as oligonucleotides could cause the immune system to produce antibodies, and that would influence the pharmacokinetic and pharmacodynamic activity of the drug.
C. Some oligonucleotides could cause blood not to clot, or to build up in the kidneys, causing kidney damage.
D. Issues such as their toxicity in the rapid expulsion of oligonucleotides from the body, chemical destruction and failure to cross cell membranes prevent their application to certain diseases (e.g., brain diseases).
E. The clinical immunogenicity testing of oligonucleotide treatments is needed to see if there are antibodies against the drug and whether those antibodies interfere with the efficacy of the drug.
F. Other oligonucleotide agents have been approved only in phase II clinical trials because of serious hematologic adverse reactions (thrombocytopenia, myelosuppression).
Moreover, there are many technical and regulatory hurdles to oligonucleotide therapies. For instance, in the nonclinical development process, toxicology packages should be developed to evaluate, where feasible, predicted toxicities of the class of treatment (targeting toxicity, immunostimulation, renal damage) and suitable species should be selected for toxicological analysis. During clinical trial, immunogenicity tests have to be taken and timed with pharmacokinetic and pharmacodynamic sampling intervals to determine if antidrug antibodies influence efficacy of oligonucleotide therapies and immune adverse events.
While oligonucleotide therapies look promising, barriers to delivery, immunogenicity, and off-target effects still astonish. Lipid nanoparticles, conjugation chemistry, and nanoparticle delivery systems have improved both the systemic spread and the cell-level absorption. Moreover, the continued advance of precision gene editing and next-generation sequencing is broadening the oligonucleotide-based tools.
Oligonucleotide therapies are still being developed, and more clinical trials are looking at novel approaches to diseases that have been thought incurable. This precision-based model is a transformation in modern medicine, with possibilities for personalised, targeted treatments like never before.
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