In recent years, small nucleic acid drugs have emerged as a focal point in biomedical research due to their strong specificity, simple design, short development cycles, and diverse target options. Small nucleic acid drugs specifically refer to a class of oligonucleotide molecules that target RNA or proteins, including antisense oligonucleotides (ASOs), small interfering RNA (siRNA) and aptamers. ASOs typically consist of 15-25 nucleotides and are short-chain nucleic acids chemically modified. They form a double-stranded structure with the target based on Watson-Crick base pairing principles. siRNA, also known as short interfering RNA or silencing RNA, is a specific-length (21-25 bp) RNA fragment produced by the host organism’s cleavage of double-stranded RNA from foreign gene expression. Since their discovery, siRNAs have been widely recognized for their ability to silence many or all genes. Currently, small nucleic acid drug delivery vehicles mainly fall into viral and non-viral categories. Non-viral carriers include GalNAc (N-acetylgalactosamine) conjugation, lipid nanoparticles (LNPs), polymers, exosomes, peptide conjugation, antibody conjugation, and various other modifications.
Classification of nucleic acid drugs
Nucleic acid drugs encompass a diverse array of therapeutic agents designed to modulate gene expression. They can be broadly classified into several categories based on their mechanisms of action and molecular structures. The primary classes include:
1. ASOs: Single-stranded DNA or RNA molecules designed to bind complementary mRNA sequences, thereby inhibiting gene expression or altering splicing.
2. siRNA: Double-stranded RNA molecules that induce RNA interference (RNAi), leading to the degradation of target mRNA and subsequent gene silencing.
3. miRNA mimics and inhibitors: Small RNA molecules that regulate gene expression post-transcriptionally, either mimicking endogenous miRNAs or inhibiting their function.
4. Ribozymes and DNAzymes: Catalytic RNA and DNA molecules that can cleave specific RNA sequences, thus modulating gene expression.
5. Aptamers: Short, single-stranded DNA or RNA molecules that can bind to specific proteins or other targets with high affinity and specificity.
Development of Small Nucleic Acid Drugs
siRNA originated in 1998 when scientists Andrew Fire and Craig Mello first revealed the phenomenon of RNA interference in the nematode worm. In 2003, several companies began developing siRNA drugs. Unfortunately, in the initial clinical trials, unmodified siRNA exhibited strong immune-related toxicity, and its efficacy was questioned. In the second round of clinical trials, the delivery of siRNA to the human body via nanocarriers confirmed its efficacy but led to severe toxic reactions and inadequate dose effectiveness. Faced with these challenges, many large companies withdrew from the field, smaller companies persevered, modifying siRNA and utilizing delivery carriers to create safer and more effective compounds. The first approved siRNA drug, Patisiran, designed for familial amyloid polyneuropathy, entered the market in 2018, marking a significant milestone.
It is evident that without delivery carriers, siRNA drugs may carry significant risks to efficacy. Mechanistic studies reveal that siRNA lacks inherent organ and tissue targeting specificity and cannot selectively act on organs. Additionally, the large molecular weight and hydrophilic nature of siRNA hinder passive membrane penetration, making distribution to target tissues challenging. Moreover, being exogenous RNAi, siRNA is prone to triggering immune reactions, making it difficult for siRNA drugs to reach target sites and achieve the required effective doses. To enhance safety and efficacy, all five approved siRNA drugs on the market have undergone specific modifications and utilize delivery carriers, primarily LNP and GalNAc, both designed to target the liver. On the other hand, the development of ASOs predates siRNA, with a more significant number of varieties already on the market. ASOs originated in 1978 when Harvard University scientists, including Zamecnik, designed and synthesized a short RNA complementary to the Rous sarcoma virus gene. They discovered that this short RNA could inhibit virus replication in cultured tissues. ASOs were first proven effective in vivo in 1993, and with various modifications and continued research, the first approved ASO product, Fomivirsen (Vitravene), emerged in 1998. Although Vitravene has been discontinued for various reasons, its significance is profound. Over time, as researchers continued to study and modify structures, an increasing number of ASO products gained approval, with a total of 10 approved products to date.
Small nucleic acid drugs examples
Several small nucleic acid drugs have been approved or are in advanced stages of clinical development, showcasing their therapeutic potential.
Patisiran (Onpattro) is an siRNA-based drug approved for the treatment of hereditary transthyretin-mediated amyloidosis. Patisiran uses lipid nanoparticles for delivery to hepatocytes, where it silences the expression of the transthyretin gene.
Nusinersen (Spinraza) is an ASO approved for the treatment of spinal muscular atrophy. Nusinersen modulates the splicing of SMN2 mRNA to increase the production of functional SMN protein.
Givosiran (Givlaari) is another siRNA therapeutic approved for the treatment of acute hepatic porphyria. It targets the ALAS1 gene in the liver to reduce the accumulation of toxic heme intermediates.
Small nucleic acid at BOC Sciences
| Name | CAT | MW |
| Nusinersen | 1258984-36-9 | 7501.0 |
| eteplirsen | 1173755-55-9 | 10305.738 |
| Trabedersen | 5768.7 | |
| Inclisiran sodium | B1370-057828 | 17,284.72 Da |
| Givosiran sodium | B1370-212888 | 17,245.56 Da |
| Lumasiran | 1834610-13-7 | 16,340 Da |
| Cemdisiran | 1639264-46-2 | 16782.53 |
| Tivanisiran | 1848224-71-4 |
Modification of Small Nucleic Acid Drugs
Nonmodified nucleic acids have limited pharmacokinetics properties and are rapidly degraded by nucleases. Modification of the nucleic acid backbone, the ribose sugar moiety and the nucleobase itself has been extensively employed in order to improve the drug-like properties of oligonucleotide drugs.
Backbone modification
The first generation of modifications focused on the backbone of its nucleic acid chain, specifically targeting the phosphorothioate (PS) bonds with the aim of improving enzyme resistance. The incorporation of PS linkages, in which one of the non-bridging oxygen atoms of the inter-nucleotide phosphate group is replaced with sulfur, is widely used in therapeutic oligonucleotides. PS backbone modifications offer several advantages:
- Thiophosphate Main Chain Modification: Extends the half-life of oligonucleotides in serum, promoting binding with serum proteins.
- Increased Cellular Uptake: Guides ribonuclease H to degrade target mRNA.
- Enhanced Enzyme Resistance: Improves resistance to enzymatic degradation.
- No Need for Delivery Carrier: Can spontaneously enter cells.
Nucleobase modification
Strategies to modify nucleobase chemistry are also being investigated. For example, pyrimidine methylation (5-methylcytidine and 5-methyluridine/ribothymidine) has the effect of increasing the oligonucleotide melting temperature by ~0.5 °C per substitution, and has been commonly incorporated into ASOs.
Ribose sugar modification
To further improve the properties of ASOs and overcome the limitations of the first generation, researchers introduced modifications at the 2′ position of the ribose, either at the ends or in the middle of the sequence. This included 2′-O-methyl (2′-OMe) and 2′-methoxyethyl (2′-MOE) modifications, offering advantages such as:
- Enhanced Drug Activity: Promotes target binding.
- Reduced Non-specific Protein Binding: Minimizes toxicity.
Bridged nucleic acids
Subsequently, more advanced third-generation structural modifications, including locked nucleic acid (LNA), constrained ethyl (cEt), and 2′-O, 4′-C-ethylene-bridged nucleic acids (ENA), were employed. As technology research progressed, newly developed chemically modified molecules often exhibited improved characteristics, including enhanced affinity with target RNA, stability against nucleases, RNAase H recognition and degradation capabilities after binding to target RNA, pharmacokinetics, pharmacodynamics, tissue distribution, half-life, and toxicity.
It is evident that ASOs with different degrees of modification can enter cells through passive diffusion, bind to serum proteins, facilitate binding with specific tissues, and exert powerful and prolonged effects even with only a small amount entering the cell nucleus. Literature reports indicate that in the cytoplasm and nucleus, only 1% to 2% of small nucleic acid drugs such as siRNA or ASOs may reach their targets. Even if less than 1% of the drug reaches the target, it can still exert a potent and persistent effect. Therefore, even without a delivery carrier, ASOs, through structural modifications alone, can enter the target cell nucleus in small amounts and exhibit therapeutic efficacy. In contrast, siRNA without a delivery carrier faces challenges in entering the cell nucleus, compromising both efficacy and safety.
Oligonucleotide modification at BOC Sciences
Delivery Modes of Small Nucleic Acid Drugs
Among the ASO products that have been marketed, there are four drugs used for Duchenne muscular dystrophy, one for familial amyloidotic polyneuropathy, two for spinal muscular atrophy, one for familial chylomicronemia syndrome, one for homozygous familial hypercholesterolemia, and one for cytomegalovirus retinitis. Among them, two are administered intrathecally, one is administered intravitreally, and the rest are administered subcutaneously or intravenously. Duchenne muscular dystrophy is primarily an X-linked recessive lethal genetic disorder, usually caused by gene mutations, and its pathogenesis is complex and diverse. The causative gene for this disease is the largest gene in humans, located in the Xp21.2 region, encoding dystrophin, a cytoskeletal protein primarily found in the cytoplasm of cardiac and skeletal muscle fibers, with the most significant distribution at neuromuscular junctions. ASO primarily employs antisense oligonucleotide-mediated exon-skipping therapy, a method that in clinical trials has shown to restore the expression function of the dystrophin gene in DMD patients. Since dystrophin is present in the cytoplasm of cardiac and skeletal muscle fibers, the drug only needs to enter peripheral tissues to be effective. Modified ASO administered through subcutaneous or intravenous injection can widely distribute to peripheral tissues.
Currently, two drugs administered intrathecally have been marketed, namely Nusinersen (Spinraza) and Tofersen (Qalsody). They are indicated for spinal muscular atrophy and amyotrophic lateral sclerosis (ALS), respectively. Spinal muscular atrophy is an autosomal recessive genetic disorder of the motor system, with an incidence rate of 1/6,000 to 1/10,000 in newborns. It is characterized by degeneration of anterior horn alpha motor neurons, leading to progressive atrophy and paralysis of proximal limbs and trunk muscles, ultimately resulting in death due to respiratory failure. ALS is a neurodegenerative disease affecting mainly the motor neurons of the cerebral cortex, brainstem, and spinal cord, with an unknown etiology. The two marketed drugs are administered intrathecally to allow the drug to act from cerebrospinal fluid to the central nervous system. Due to its high charge, ASO cannot pass the blood-brain barrier. To enable the drug to enter the central nervous system, there are four administration methods: systemic administration (via nanodelivery), intrathecal injection, intraventricular injection, and nasal delivery. Research on treating Huntington’s disease by inhibiting huntingtin protein has revealed that through intrathecal injection of ASO, the drug can widely distribute in the brain and effectively suppress huntingtin protein. This indicates that intrathecal injection allows targeted action of ASO on the central nervous system.
Development of Delivery Carriers
The delivery potential of ASOs and siRNAs can be enhanced through direct covalent conjugation of various moieties that promote intracellular uptake, target the drug to specific cells/tissues or reduce clearance from the circulation. These include liposomes, GalNAc modifications, antibodies, peptides, copolymers, etc.
Lipid conjugates
Covalent conjugation to lipid molecules has been used to enhance the delivery of siRNAs and antagomir ASOs. Cholesterol siRNAs (conjugated to the 3ʹ terminus of the passenger strand) have been utilized for hepatic gene silencing and, more recently, to silence myostatin (Mstn) in murine skeletal muscle (a target organ in which it has historically been particularly challenging to achieve effective RNAi) after systemic delivery. In vivo association of siRNAs with the different classes of lipoprotein is governed by their overall hydrophobicity, with the more hydrophobic conjugates preferentially binding to LDL and the less lipophilic conjugates preferentially binding to HDL.
GalNAc conjugates
GalNAc is a carbohydrate moiety that binds to the highly liver-expressed asialoglycoprotein receptor 1 (ASGR1, ASPGR) with high affinity and facilitates the uptake of PO ASOs and siRNAs into hepatocytes by endocytosis. GalNAc conjugation enhanced ASO potency by ~7-fold in mouse, specific to the liver, and by ~30-fold in human patients. As such, GalNAc conjugation is now one of the leading strategies for delivering experimental oligonucleotide drugs currently in development, given its high liver silencing potential, small size relative to nanoparticle complexes, defined chemical composition and low cost of synthesis.
Antibody and aptamer conjugates
Specific interactions between an antibody and a cell surface receptor have the potential to enable delivery to tissues and/or cell subpopulations that are not accessible using other technologies. Various receptors have been successfully targeted for siRNA delivery, including the HIV gp protein, HER2, CD7, CD71 and TMEFF2. Similarly, ASOs have also been conjugated with antibodies against CD44, EPHA2 and EGFR.
The conjugation of therapeutic oligonucleotides to nucleic acid aptamers has also been explored for enhancing delivery of siRNAs and ASOs to specific target cells. Aptamers can be considered ‘chemical antibodies’ that bind to their respective target proteins with high affinity, but present numerous advantages over antibodies as they are simple and inexpensive to manufacture, are smaller in size and exhibit lower immunogenicity
Peptide conjugates
Peptides are an attractive source of ligands that may confer tissue/cell-targeting, cell-penetrating (that is, CPPs) or endosomolytic properties onto therapeutic oligonucleotide conjugates. CPPs are short amphipathic or cationic peptide fragments that are typically derived from naturally occurring protein translocation motifs or are based on polymers of basic amino acids. One of the most promising applications of CPPs is their direct chemical conjugation to charge-neutral ASO chemistries, such as PMO and PNA.
Lipoplexes and liposomes
Formulation with lipids is one of the most common approaches to enhancing nucleic acid delivery. Mixing polyanionic nucleic acid drugs with lipids leads to the condensing of nucleic acids into nanoparticles that have a more favourable surface charge, and are sufficiently large (~100 nm in diameter) to trigger uptake by endocytosis. Lipoplexes are the result of direct electrostatic interaction between polyanionic nucleic acid and the cationic lipid, and are typically a heterogeneous population of relatively unstable complexes. Lipoplex formulations need to be prepared shortly before use, and have been successfully used for local delivery applications. By contrast, liposomes comprise a lipid bilayer, with the nucleic acid drug residing in the encapsulated aqueous space. Liposomes are more complex (typically consisting of cationic or fusogenic lipids (to promote endosomal escape) and cholesterol PEGylated lipid) and exhibit more consistent physical properties with greater stability than lipoplexes.
References
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2. M, M, Evers.; et al. Antisense Oligonucleotides: Treating Neurodegeneration at the Level of RNA. Neurotherapeutics. 2013, 10(3): 486–497.
3. Roberts, T. C.; et al. Advances in oligonucleotide drug delivery. Nature reviews Drug discovery. 2020, 19(10): 673-694.