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.
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.
Modification of Small Nucleic Acid Drugs
In order to enhance the stability and biological activity of ASOs, different degrees of structural modifications have been applied. 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. 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.
However, the first-generation modifications also have drawbacks, including a potential decrease in target binding affinity. 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.
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.
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
Although the marketed ASOs products currently do not utilize specific delivery carriers, relying solely on structural modifications, it does not imply that ASOs do not benefit from delivery carriers altogether. With advancing understanding of the cellular uptake mechanisms of ASOs and the evolution of delivery methods, some ASOs now incorporate delivery carriers such as liposomes, GalNAc modifications, antibodies, peptides, copolymers, etc. However, the majority of these products are still in the clinical research phase, and certain delivery carriers may exhibit more pronounced advantages in the application of siRNA.
According to the research by Thazha P, ASOs linked with GalNAc have been found to effectively deliver ASOs to liver cells, enhancing their activity by tenfold. In incomplete statistics, there are over 50 ASO drugs globally in various stages of clinical development, with some utilizing GalNAc conjugation.
In summary, the currently marketed ASOs do not utilize delivery systems primarily because:
- Modified ASOs can adsorb onto plasma proteins, facilitating their binding to cells and specific tissue absorption.
- Some therapeutic drugs for certain diseases can exert their effects by entering peripheral tissues.
- Desired effects can be achieved through appropriate administration routes.
- Some ASO drugs utilizing delivery carriers are still in the clinical research phase.
In contrast, the majority of siRNA products have undergone delivery carrier systems, mainly because siRNA cannot bind with plasma proteins and enter target cells spontaneously. Therefore, delivery carriers are necessary to precisely transport siRNA to target organs, enhancing the effectiveness and safety of nucleic acid drugs.
References
1. Y, K, Kim. RNA therapy: Rich History, Various Applications and Unlimited Future Prospects. Experimental & Molecular Medicine. 2022, 54: 455-465.
2. M, M, Evers.; et al. Antisense Oligonucleotides: Treating Neurodegeneration at the Level of RNA. Neurotherapeutics. 2013, 10(3): 486–497.