As a vital drug modality, the field of oligonucleotide development will continue to expand and strengthen in the coming years. Oligonucleotide therapeutics are a class of medicines composed of short nucleotide sequences (components of DNA and RNA) that have advanced new mechanisms for controlling gene expression. By modulating gene expression levels, these drugs inhibit the production of pathogenic proteins.
Oligonucleotide Drug Development Process
1. Target Identification
Oligonucleotide drugs are designed to target specific genes, messenger RNA (mRNA), or non-coding RNA (e.g., microRNA) involved in disease processes. Target identification is a critical step in developing these drugs.
2. Sequence Design
Oligonucleotide drugs are inherently designed based on the target RNA, with their nucleotide sequences complementary to the target RNA. This provides them with high binding specificity.
3. Binding to Target RNA
Once inside the body, oligonucleotide drugs circulate in the bloodstream and selectively bind to target RNA molecules through complementary base pairing. This binding can occur through multiple mechanisms:
- mRNA Degradation: Some oligonucleotide drugs trigger the degradation of target mRNA. Upon binding, they recruit cellular enzymes to degrade the RNA molecule, preventing its translation into proteins.
- Translation Blockage: Certain oligonucleotide drugs physically obstruct ribosome binding sites or interfere with other translation processes, blocking the translation of mRNA into proteins.
- Splicing Modulation: When the target is precursor mRNA (pre-mRNA), these drugs can influence alternative splicing. By binding to specific splice sites, they can promote the inclusion or exclusion of specific exons, leading to changes in protein isoforms.
- RNA Interference (RNAi): Some oligonucleotide drugs function through RNA interference mechanisms. They are integrated into the RNA-induced silencing complex (RISC), guiding it to the target mRNA, resulting in its degradation or translation suppression.
4. Impact on Protein Production
By regulating target RNA, oligonucleotide drugs can reduce the production levels of pathogenic proteins. They are often used to treat various genetic and rare diseases caused by protein overproduction or dysfunction.
It is noteworthy that oligonucleotide therapeutics demonstrate promising potential in treating various diseases, including genetic disorders, neurodegenerative diseases, and certain types of cancer. They are especially impactful in addressing many traditionally “undruggable” targets. However, their development can be complex and may involve limitations and potential side effects that require careful consideration in clinical applications.
Antisense Oligonucleotides (ASOs)
Antisense oligonucleotides (ASOs) are chemically modified single-stranded fragments of DNA or RNA, typically 15-30 nucleotides in length. They specifically bind to endogenous mRNA targets through Watson-Crick base pairing rules. ASOs encompass both antisense RNA (asRNA) and antisense DNA (asDNA).
From a drug-like property perspective, chemical modifications are commonly applied to ASOs to ensure stability under physiological conditions and to enhance their efficacy in inhibiting mRNA targets.
Mechanisms of Action of ASOs
ASOs exert their therapeutic effects through various mechanisms, including:
- Translation Inhibition: Blocking the production of proteins by interfering with ribosome assembly or translation machinery.
- mRNA Degradation: Triggering the breakdown of target mRNA, often via the recruitment of RNase H enzymes.
- Splicing Modulation: Altering the splicing of precursor mRNA (pre-mRNA) to produce specific protein isoforms.
These mechanisms allow ASOs to be used in gene silencing applications and the treatment of genetic disorders, cancer, and neurodegenerative diseases.
Phosphorothioate Oligonucleotides
Phosphorothioates represent one of the earliest backbone modifications for oligonucleotide drugs. Fomivirsen (brand name Vitravene) was the first ASO approved for human use. It is an antiviral phosphorothioate oligonucleotide derivative used to treat cytomegalovirus-induced retinitis, a condition that can lead to blindness in AIDS patients without intervention.
Benefits of Phosphorothioate Backbone Modifications
Extended Half-life: Improves ASO stability in serum.
Enhanced Protein Binding: Facilitates ASO transport and distribution in the bloodstream.
Increased Cellular Uptake: Promotes the degradation of target mRNA via RNase H activation.
These advancements underscore the therapeutic significance of phosphorothioate oligonucleotides in the development of ASO-based medicines.
Aptamers
Aptamers can be single-stranded DNA, RNA molecules, or peptide molecules with unique three-dimensional structures. These structures allow aptamers to selectively bind specific target molecules, such as proteins, nucleic acids, small molecules, and even cells. Sometimes referred to as “chemical antibodies,” aptamers exhibit high specificity and affinity in recognizing and binding to their targets. Compared to antibodies, aptamers offer several advantages, including smaller size, ease of synthesis, and potential for chemical modifications. Additionally, they generally do not elicit immune responses when used in vivo.
Aptamers derived from oligonucleotides are non-toxic and non-immunogenic single-stranded DNA or RNA molecules, typically 20 to 80 nucleotides in length. These aptamers can achieve binding affinities comparable to antibodies and can be synthetically produced and chemically modified on a large scale to optimize their pharmacokinetic properties.
Pegaptanib (brand name Macugen) is the only FDA-approved aptamer for human treatment to date (several other aptamers are in clinical trials). Pegaptanib is a chemically conjugated oligonucleotide linked to polyethylene glycol (PEG). It was approved by the FDA in December 2004 for the treatment of neovascular (wet) age-related macular degeneration (AMD).
The ability to synthesize aptamers with tailored properties highlights their potential as a versatile platform for therapeutic and diagnostic applications.
Pegaptanib demonstrates high affinity for vascular endothelial growth factor (VEGF), specifically binding to the VEGF-165 isoform. VEGF-165 is a critical protein involved in angiogenesis and increased vascular permeability (vascular leakage), which are two major pathological processes of age-related macular degeneration (AMD). Pegaptanib acts as a VEGF antagonist, blocking VEGF activity when injected into the eye. This inhibition reduces abnormal blood vessel growth and controls leakage and swelling, providing therapeutic benefits for AMD patients.
Gapmer
Gapmer technology represents the second generation of antisense oligonucleotide (ASO) therapeutics. Gapmers are short DNA or RNA antisense oligonucleotides that incorporate 2′-O-alkyl (or other chemical modifications) into their structure. These modifications enhance the stability of antisense oligonucleotides against nucleases. However, such modifications typically prevent recognition by RNase H, an enzyme critical for the cleavage of RNA strands in RNA-DNA hybrids.
Gapmer technology addresses this limitation by designing a central unmodified DNA segment flanked by modified regions. The central unmodified region retains high affinity for RNase H, enabling it to recruit the enzyme for RNA cleavage. Meanwhile, the modified flanking regions improve resistance to nuclease degradation and increase binding affinity and biological activity.
Mechanism of Action
Gapmers hybridize with target RNA fragments and silence the corresponding gene by recruiting RNase H to degrade the target RNA through enzymatic cleavage. The chemically modified flanking regions enhance stability, allowing gapmers to bind their targets with higher affinity and retain robust bioactivity.
Gapmers are under development for treating a variety of conditions, including cancers, viral infections, and chronic genetic disorders. The dual benefits of enhanced stability and gene silencing efficiency position gapmers as a promising technology in the field of oligonucleotide therapeutics.
Chemical Modifications in Gapmers
Gapmers incorporate chemical modifications that significantly enhance their stability against nuclease degradation. These modifications include LNA (locked nucleic acids), 2′-OMe (2′-O-methyl), and 2′-F (2′-fluoro) groups, creating chemical analogs of natural RNA. These chemical modifications confer several benefits:
- Enhanced Nuclease Resistance: The modifications protect the gapmer oligonucleotides from enzymatic degradation in biological systems, prolonging their functional half-life.
- Reduced Immunogenicity and Toxicity: Modifications such as 2′-OMe and 2′-F reduce the likelihood of triggering immune responses and decrease the overall cytotoxicity of the therapeutic molecule.
- Improved Target Affinity: The chemically modified bases allow gapmers to bind their target mRNA with higher specificity and affinity. This high affinity minimizes off-target effects, non-specific interactions, and unintended gene silencing.
Mipomersen (Kynamro)
Mipomersen, marketed under the name Kynamro, is a 20-base gapmer designed to inhibit apolipoprotein B. It was approved for use in 2013. Mipomersen is used to treat hyperlipidemia by lowering low-density lipoprotein (LDL) cholesterol levels.
The structure of Mipomersen features thioate linkages between nucleotides rather than the traditional phosphodiester bonds. Additionally, the sugar moiety of the molecule is modified with 2′-O-methoxyethyl (2′-OMe) groups. These modifications confer several key properties:
- Resistance to Nuclease Degradation: The thioate modifications and 2′-OMe sugar modifications protect Mipomersen from enzymatic degradation, allowing the drug to maintain stability in the body.
- Longer Dosing Intervals: The chemical modifications enable weekly dosing, which is a significant advantage for patient compliance and convenience.
Exon Skipping with RNA Splicing Modulators
Another critical area for antisense oligonucleotides (ASOs) is RNA splicing modulation, specifically exon skipping, which can be used to treat diseases caused by genetic mutations that disrupt normal splicing of mRNA. This strategy involves designing ASOs to bind to specific regions of pre-mRNA to either exclude or include certain exons during splicing.
Exon Skipping: This process skips the faulty exon, restoring the reading frame of the gene and allowing the production of a functional, albeit truncated, protein. In diseases like Duchenne muscular dystrophy (DMD), exon skipping helps bypass mutated exons to produce a shortened but functional version of the dystrophin protein, which is essential for muscle function.
Key Drugs in Exon Skipping:
Eteplirsen (Exondys 51): Approved for DMD, this PMO-based ASO skips exon 51 of the dystrophin gene, allowing the production of a functional dystrophin protein.
Nusinersen (Spinraza): Used in the treatment of spinal muscular atrophy (SMA), it targets the splicing of the SMN2 gene, producing more of the SMN protein that is deficient in SMA patients.
Golodirsen (Vyondys 53): A gapmer targeting exon 53 of the dystrophin gene for DMD treatment.
Vitolarsen (Viltepso): Another gapmer for DMD, targeting exon 53.
Casimersen (Amondys 45): Targets exon 45 of the dystrophin gene for DMD treatment.
Phosphorodiamidate Morpholino Oligomers (PMO)
PMOs represent another class of ASO modifications, playing a significant role in exon skipping. PMOs are synthetic nucleic acid analogs that use morpholino rings instead of sugar-phosphate backbones, making them neutral and resistant to nucleases. This allows them to be more stable and to effectively bind to target mRNA to block protein translation.
Notable PMO-based Drug:
Eteplirsen (Exondys 51): A milestone in PMO-based therapies, this drug has been approved for treating Duchenne muscular dystrophy (DMD) by promoting exon skipping in the dystrophin gene, restoring the expression of a functional, albeit shorter, dystrophin protein.
Oligo Modification Services at BOC Sciences
- Oligo Backbone Modification
- Oligo Base Modification
- Oligo splicing and chemical modification
- Oligo Spacer Modification
- Oligo Phosphorylation Modification
- Oligo Phosphorylation Modification
- Backbone Modifications
- 2′-Modifications
- Labeling Modifications
- Fluorescent Modifications
RNA Interference (RNAi) and herapeutic siRNA
RNA interference (RNAi) is a natural cellular process that regulates gene expression by degrading mRNA, preventing its translation into protein. It plays a critical role in controlling gene activity and protecting cells from viruses. RNAi occurs through small double-stranded RNA molecules that can be classified into several types, including:
- siRNA (small interfering RNA): Primarily involved in gene silencing by degrading target mRNA.
- miRNA (microRNA): Regulates gene expression at the post-transcriptional level by binding to complementary sequences on target mRNA.
- piRNA (Piwi-interacting RNA): Involved in silencing transposons and maintaining genome stability in germline cells.
Therapeutic applications of RNAi involve the delivery of synthetic small RNA molecules (e.g., siRNA, artificial miRNA, or shRNA) to targeted cells to induce gene silencing. These molecules engage the RNA-induced silencing complex (RISC), which leads to the degradation of the target mRNA, preventing protein synthesis and thereby halting the progression of disease.
RNAi can also be used to combat viral infections by targeting viral mRNA or even genomic RNA, thereby preventing the virus from replicating within the host cells.
Oligonucleotide Drug Delivery
Like many macromolecular drugs, oligonucleotide therapies face a significant challenge in drug delivery, particularly for RNA-based drugs, due to their typically poor cellular uptake and low stability under physiological conditions.
Liposome formulations can address this issue, or conjugates containing recognition molecules can be prepared. For example, Patisiran (Onpattro), used for the treatment of hereditary transthyretin-mediated amyloidosis causing polyneuropathy. Patisiran is the first siRNA drug approved for this indication, delivering the drug directly to the liver using lipid nanoparticles (LNPs).
The approval of Givosiran (Givlaari) for the treatment of hereditary liver porphyria was a landmark event, as it was the first siRNA drug actively transported to the liver using N-acetylgalactosamine. The siRNA molecule inhibits 5-aminolevulinate synthase. This formulation is administered via subcutaneous injection, causing the siRNA to accumulate in target liver cells. Its success has led to the development of several other siRNA drug delivery pathways, such as Inclisiran (Leqvio), Lumasiran (Oxlumo), and Vutrisiran (Amvuttra).
Advancements in Oligonucleotide-Based Therapies
Oligonucleotide-based therapies are making significant strides, with ongoing research and development focused on discovering and developing new targets and mechanisms of action. One promising area is the use of oligonucleotides as microRNA (miRNA) regulators, a strategy that has the potential to address a variety of diseases associated with miRNA dysregulation, such as cancer, neurodegenerative diseases, and autoimmune disorders.
miRNA-based therapies can be broadly categorized into two main approaches:
- miRNA Mimics (Agonists, dsRNA): These are used to restore suppressed miRNA levels. By introducing synthetic miRNA mimics, it’s possible to restore the function of miRNAs that may be underexpressed in certain disease states.
- Endogenous miRNA Inhibitors (Antagonists, ssRNA): Known as antimiRNA or antagomir, these oligonucleotides downregulate overactive miRNAs. By blocking the activity of these miRNAs, these therapies can potentially reverse the effects of diseases where miRNAs are upregulated or dysregulated.
A significant advancement in oligonucleotide drug delivery involves the use of GalNAc (N-acetylgalactosamine) conjugates, which have successfully targeted liver cells, leading to a surge in research interest in this field. GalNAc conjugation allows oligonucleotides to be selectively delivered to the liver, a key organ for many therapeutic interventions.
Additionally, there is growing interest in developing novel oligonucleotide conjugates for non-liver delivery, especially focusing on peptide-oligonucleotide and lipid-oligonucleotide conjugates. These conjugates can facilitate cellular uptake through receptor-mediated mechanisms and can be combined with small molecules to enhance tissue targeting.
Another innovative approach involves the use of PROTACs (Proteolysis Targeting Chimeras), which can induce protein degradation pathways, offering the possibility of controlling protein levels in disease processes.
Challenges in CMC (Chemistry, Manufacturing, and Controls)
Despite the progress, the oligonucleotide industry faces an important challenge in the CMC field, particularly in terms of reducing reagent and solvent consumption during the production process. The green chemistry approach to optimizing production processes is crucial for reducing environmental impact and improving the sustainability of oligonucleotide drug manufacturing.
In the coming years, the optimization of manufacturing processes to make them more efficient and environmentally friendly will be a key focus area for the oligonucleotide industry. Ensuring that production scales efficiently while minimizing waste and resource consumption will be essential for the future success of oligonucleotide-based therapies.
Summary
The oligonucleotide therapeutics field is advancing rapidly, with a focus on miRNA regulation, novel delivery technologies, and sustainable manufacturing. Key innovations such as GalNAc conjugates, PROTACs, aptamers, and DNA nanotechnology are paving the way for more effective and targeted treatments. As the industry addresses challenges in drug delivery and manufacturing efficiency, the potential for oligonucleotides to treat a wide range of diseases continues to grow.