As an important drug modality, the development of oligonucleotides will continue to expand in the coming years. Oligonucleotide Drugs are a class of therapeutic agents composed of short nucleotide sequences (components of DNA and RNA). Their advancements have led to new mechanisms of gene expression control, enabling the regulation of gene expression levels and the inhibition of the production of disease-causing proteins through drug intervention.
Oligonucleotide Drug Development Process:
01 Target Identification
Oligonucleotide Drugs target specific genes, messenger RNA (mRNA), or non-coding RNA (e.g., microRNA) involved in disease processes. Target identification is a crucial step in the development of these drugs.
02 Sequence Design
Oligonucleotide drugs are fundamentally designed based on the target RNA, with their nucleotide sequence complementary to the target RNA. This imparts them with high binding specificity.
03 Binding to Target RNA
- Once inside the body, oligonucleotide drugs circulate in the bloodstream and selectively bind to the target RNA molecules through complementary base pairing. This affinity can occur through various mechanisms:
- mRNA degradation: Some oligonucleotide drugs can trigger the degradation of the target mRNA. When these drugs bind to the mRNA, they can recruit cellular enzymes to degrade the RNA molecule, preventing its translation into protein.
- Translation inhibition: Certain oligonucleotide drugs can physically obstruct the ribosome binding site or interfere with other translation processes, thus blocking mRNA translation into protein.
- Splicing regulation: If the target of the oligonucleotide drug is precursor mRNA, these drugs can influence alternative splicing. By binding to specific splicing sites, they can promote the inclusion or exclusion of specific exons, leading to changes in protein isoforms.
- RNA interference (RNAi): Some oligonucleotide drugs can exert their effects through the RNA interference mechanism. They integrate into the RNA-induced silencing complex (RISC) and guide RISC to locate the target mRNA, leading to its degradation or translation inhibition.
04 Impact on Protein Production
By regulating target RNA, oligonucleotide drugs can reduce the production levels of disease-causing proteins. They are typically used to treat various genetic and rare diseases caused by overproduction or dysfunction of proteins.
It is worth noting that oligonucleotide drugs show promising prospects in the treatment of various diseases, including genetic diseases, neurodegenerative diseases, and certain types of cancers, especially in targets traditionally considered “undruggable.” However, their development can be complex, and they may have limitations and potential side effects that need to be carefully considered in clinical use.
Antisense Oligonucleotides
Antisense oligonucleotides (ASOs) are chemically modified single-stranded DNA or RNA fragments, typically consisting of 15-30 nucleotides. They specifically bind to endogenous mRNA targets through Watson-Crick base-pairing rules. ASOs include both antisense RNA (asRNA) and antisense DNA (asDNA). From a pharmacological perspective, chemical modifications are often necessary for ASOs to ensure stability under physiological conditions and enhance their efficacy in inhibiting mRNA targets. The mechanisms of action of ASOs include translation inhibition, mRNA degradation, and splicing regulation. They can be applied in gene silencing and the treatment of certain genetic diseases, cancer, and neurodegenerative diseases. ASOs have shown promise in the treatment of conditions like spinal muscular atrophy (SMA), Duchenne muscular dystrophy (DMD), amyotrophic lateral sclerosis (ALS), and certain cancers. For example, nusinersen (Spinraza) is an ASO approved for the treatment of SMA.
Phosphorothioate Oligonucleotides
Phosphorothioates are the original backbone modification of oligonucleotide drugs. Fomivirsen is the first antisense oligonucleotide drug approved for human therapeutic use. It is an antiviral agent of a phosphorothioate oligonucleotide derivative that treats cytomegalovirus-induced retinitis, a disease that occurs in some patients with AIDS and can lead to blindness if not treated with intervention. The main chain modification of the phosphorothioate extends the half-life of the oligonucleotide in serum, promotes its binding to serum proteins, increases cellular uptake, and directs ribonuclease H to degrade the target mRNA.
Aptamer
Aptamers can be single-stranded DNA, RNA molecules, or even peptide molecules that possess unique three-dimensional structures. They have the ability to selectively bind to specific target molecules, such as proteins, nucleic acids, small molecules, and even cells. Aptamers are sometimes referred to as “chemical antibodies” because they can recognize and bind to their targets with high specificity and affinity. Compared to antibodies, aptamers have several advantages, including smaller size, ease of synthesis, and modification potential. When used in the body, they typically do not elicit an immune response. In the case of oligonucleotides, aptamers are non-toxic and non-immunogenic single-stranded DNA or RNA oligonucleotides with lengths ranging from 20 to 80 nucleotides. These aptamer molecules may have binding affinities similar to antibodies and can be synthesized and modified on a large scale to optimize pharmacokinetic properties. Pegaptanib is currently the only aptamer approved for human therapy (several aptamers are undergoing clinical trials). Pegaptanib consists of oligonucleotides chemically linked to polyethylene glycol (PEG) and is used to treat wet age-related macular degeneration (AMD). It received FDA approval in December 2004.
Pegaptanib has high affinity for vascular endothelial growth factor (VEGF). It specifically binds to the 165 subtype of VEGF, which is a protein that plays a critical role in angiogenesis and increased permeability (vascular leakage), two major pathological processes in AMD. Pegaptanib is an antagonist of VEGF, and when injected into the eye, it inhibits the action of VEGF, reducing the growth of intraocular blood vessels and helping to control leakage and swelling.
Gapmer
Gapmer technology represents the second generation of oligonucleotide drugs. Gapmers are short DNA or RNA antisense oligonucleotides that introduce 2′-O-alkyl (and other modifications) structures into the oligonucleotide molecules. These modifications increase the stability of antisense oligonucleotides against nucleases, but these derivatives are not recognized by ribonuclease H (RNAse-H enzymes). This issue can be addressed through Gapmer technology: the central portion of the Gapmer molecule is not modified with 2′-O-alkyl (or other modified groups), allowing it to retain a high affinity for RNAse-H enzymes, while its flanking ends can be modified with 2′-O-alkyl groups. Gapmers hybridize with the target RNA segment and silence the target gene through the recruitment of RNAse-H enzymes via their cleavage activity. The modified RNA flanking regions make them more resistant to nuclease degradation, resulting in higher affinity and biological activity in the binding of Gapmers to the target. Gapmers are currently being developed for the treatment of various cancers, viruses, and other chronic genetic diseases. Chemical modifications in Gapmers increase their resistance to nuclease degradation. Bases modified with LNA, 2′-OMe, or 2′-F derivatives create chemical analogs of natural RNA. These modifications enhance nuclease resistance, reduce immunogenicity, and lower toxicity. Gapmers can also achieve higher affinity for the target mRNA, reducing off-target effects, non-specific binding, and unwanted gene silencing. Mipomersen is a 20-base Gapmer that inhibits apolipoprotein B and was approved in 2013. Mipomersen treats hyperlipidemia by lowering low-density lipoprotein (LDL) levels. In the Mipomersen molecule, nucleotides are linked by a thio-phosphorothioate bond, instead of the traditional phosphodiester bond, and the sugar portion is modified with 2′-O-methoxyethyl. These modifications enable the drug to resist nuclease degradation, allowing for a once-weekly dosing frequency.
RNA Splicing Modulating Antisense Oligonucleotides for Exon Skipping
Antisense oligonucleotides (ASOs) can also be designed with sequences complementary to splicing regions to treat genetic diseases. This strategy can be used to treat diseases with splicing defects, allowing for the induction of exon exclusion or inclusion. In molecular biology, exon skipping is a form of RNA splicing used to “skip” erroneous exon segments in the genetic code. Although exon skipping results in gene alterations, the modified gene can still produce truncated proteins, which can retain their biological functions. Genes consist of introns and exons. Exons are parts of the DNA that contain the instructions for protein production. Interspersed within these segments are non-coding regions called introns. Before protein manufacturing, introns are removed, leaving behind the coding exonic regions. For those exons that result in genetic mutations, ASOs can be used to induce exon skipping by binding to the mutation site in the precursor RNA. ASOs bind to the mutated exon, so when the gene is translated from mature mRNA, it gets “skipped,” thereby restoring the disrupted gene reading frame. This process generates internally deleted but still predominantly functional proteins. This mechanism is used to treat diseases like Duchenne muscular dystrophy (DMD).
ASO drugs in this field include Eteplirsen (Exondys 51), Nusinersen (Spinraza), Golodirsen (Vyondys 53), Vitolarsen (Viltepso), and Casimersen (Amondys 45). These ASOs undergo chemical modifications involving phosphorothioate bonds and 2′-O-alkyl RNA, particularly 2′-O-methoxyethyl (MOE) modifications. Phosphorodiamidate morpholino oligomers (PMOs) are a significant ASO modification. PMOs are synthetic DNA analogs capable of suppressing gene expression. The backbone of PMOs consists of morpholine rings linked by phosphorodiamidate bonds. As neutrally charged nucleic acid analogs, PMOs bind to complementary sequences in the target mRNA via Watson-Crick base pairing, blocking protein translation through steric hindrance. PMOs exhibit resistance to various enzymes present in biological fluids. Notably, Eteplirsen, a therapy for Duchenne muscular dystrophy (DMD) based on PMOs, has received FDA approval, marking a milestone event in the field of exon-skipping antisense therapy.
RNA Interference and Therapeutic siRNA
RNA interference is a natural regulatory process that downregulates gene expression through small double-stranded RNA (dsRNA). It is typically categorized into three types:
- Small Interfering RNA (siRNA)
- MicroRNA (miRNA)
- Piwi-Interacting RNA (piRNA)
Therapies based on RNAi involve the delivery of synthetic small RNA duplexes (such as siRNA, artificial miRNA, and short hairpin RNA (shRNA)) into specific target cells, resulting in gene silencing effects. These small RNA molecules associate with and activate protein complexes, especially the RNA-induced silencing complex (RISC). Once bound, they can interact with the target mRNA and physically block the ribosome from continuing the synthesis of the relevant protein, marking the mRNA for degradation. RNA interference can lead to the destruction of the mRNA before its natural degradation, thereby interrupting the disease pathway. RNA interference can also protect cells from viral invasion by attacking cellular mRNA before the cellular machinery can produce viral proteins, and sometimes it even targets the viral genome itself, thereby shielding cells from viral harm.
Oligonucleotide Drug Delivery
Like many large molecule therapies, oligonucleotide therapies face a significant challenge in drug delivery, especially RNA-based drug delivery, because their cellular uptake is often inefficient, and their stability under physiological conditions is low. This challenge can be addressed by using lipid formulations or by preparing complexes with recognition molecules. For example, Patisiran (Onpattro) targets hereditary transthyretin-mediated amyloidosis, a multiple systemic neuropathy. Patisiran is the first siRNA drug approved for this indication and delivers the drug directly to the liver using lipid nanoparticles (LNPs). The approval of Givosiran (Givlaari) for treating acute hepatic porphyria in humans is a significant milestone because it represents the first use of N-acetylgalactosamine trimer (GalNAc) for actively transporting siRNA drugs to the liver, where the siRNA molecules inhibit aminolevulinic acid synthase. This formulation is administered via subcutaneous injection, allowing the siRNA to accumulate in target liver cells. Its success has paved the way for several other siRNA drug delivery pathways, including Inclisiran (Leqvio), Lumasiran (Oxlumo), and Vutrisiran or Amvuttra.
One of the areas in the quest for new drugs involves the use of oligonucleotides as regulators of microRNAs. Dysregulation of miRNAs is linked to many human diseases, including cancer, neurodegenerative disorders, and autoimmune conditions. Synthetic miRNAs hold substantial promise in the realm of modulating endogenous miRNA expression. miRNA therapy can be categorized into two major strategies:
- miRNA mimics (agonists, dsRNA): Used to restore suppressed miRNA levels.
- Endogenous miRNA inhibitors (antagonists, ssRNA), often referred to as antimiRNA or antagomirs, are employed to downregulate overactive miRNA expression.
With GalNAc as a delivery vehicle successfully targeting oligonucleotide drugs to hepatocytes, this field has gained significant momentum. A hotspot within this field involves the development of novel oligonucleotide conjugates for extracellular delivery, particularly peptide-oligonucleotide and lipid-oligonucleotide hybrids. Novel entities formed by combining small molecules with oligonucleotides are also of paramount importance. These small molecules can enhance cellular uptake through receptor-mediated mechanisms and induce protein degradation pathways via PROTAC (Proteolysis Targeting Chimeras). Aptamers represent another pivotal direction in the development of oligonucleotide drugs.
DNA nanotechnology is yet another promising field that can provide intricate nanostructures for therapy and diagnosis, thereby improving the pharmacokinetics and pharmacodynamics of oligonucleotide drugs.
Lastly, the oligonucleotide industry faces a significant challenge in the Chemistry, Manufacturing, and Controls (CMC) domain. The emphasis is on producing oligonucleotide drugs with reduced reagent and solvent consumption under more environmentally friendly conditions. This will be a primary focus in the oligonucleotide industry’s CMC efforts over the coming years.
References
2. S, Aguti.; et al. Exon-Skipping Oligonucleotides Restore Functional Collagen VI by Correcting a Common COL6A1 Mutation in Ullrich CMD. Cell. 2020, 21: 205-216.
3. R, L, Juliano.; et al. The Delivery of Therapeutic Oligonucleotides. Nucleic Acids Research. 2016, 44(14).
