Oligonucleotide drugs are short sequences of nucleotides designed to interact with specific target molecules within cellular pathways. These drugs include antisense oligonucleotides, small interfering RNAs, microRNA, and aptamers. They regulate gene expression through various mechanisms and interfere with disease-related molecular processes.

Oligonucleotide drugs differ significantly from traditional drugs in terms of design, targeting, and mechanisms of action. Traditional drugs mainly target proteins or other cellular components, while oligonucleotide drugs regulate gene expression at the genetic or molecular level by interacting with RNA or DNA. The development of oligonucleotide drugs usually involves designing sequences that specifically target particular genes, bypassing the blind spots often encountered in traditional small molecule drug discovery. This results in faster development timelines, lower costs, and higher specificity. Advances in chemical modifications and delivery systems have greatly improved the specificity and efficacy of oligonucleotide drugs while reducing side effects, making them promising tools for treating various diseases.

Types and Mechanisms of Oligonucleotide Drugs

Oligonucleotide drugs encompass a variety of molecules, each with unique structural features and mechanisms of action.

Antisense Oligonucleotides (ASOs)

ASOs are short, single-stranded nucleic acid sequences that selectively bind to target RNA through Watson-Crick base pairing. ASOs can induce the degradation of target mRNA by activating RNase H, thereby reducing the expression of the target protein. Alternatively, they can bind to the 5' untranslated region (UTR) or the translation initiation site of mRNA, creating a steric hindrance that prevents ribosome binding or movement, thereby inhibiting protein translation. ASOs can also bind to splice sites on pre-mRNA, altering exon inclusion or skipping to produce different mRNA splice variants, thereby correcting or disrupting gene expression.

Figure 1. Schematic illustrations of ASOs. (Miao et al., 2024)

RNA Interference (RNAi)

RNA interference involves the introduction of double-stranded RNA (dsRNA) to specifically degrade mRNA, leading to gene silencing. siRNAs are double-stranded RNA molecules, 20-25 base pairs in length. After unwinding, single-stranded siRNA is incorporated into the RNA-induced silencing complex (RISC). The siRNA guides RISC to the complementary sequence on the target mRNA, activating the endonuclease within RISC to cleave the mRNA, leading to mRNA degradation and gene silencing.

miRNAs (microRNAs) are key molecules in nucleic acid-based therapeutics due to their crucial roles in post-transcriptional gene regulation. These small, single-stranded RNAs, typically 21 to 23 nucleotides in length, regulate gene expression by binding to specific mRNA targets with partial complementarity, influencing both protein synthesis and gene transcription. Unlike highly specific siRNAs, which require near-perfect complementarity to their target mRNA for effective cleavage, miRNAs can target multiple genes, thus playing a broader role in gene regulation. miRNAs are initially transcribed as primary miRNA (pri-miRNA) by RNA polymerase II, then processed by Drosha and Dicer into mature miRNAs, which are incorporated into the RISC complex. Currently, miRNA-based therapeutics include miRNA mimics, which enhance the regulatory effects of endogenous miRNAs, and antimiRs, which inhibit miRNA function by competitively binding to target miRNAs. These therapeutic approaches hold significant potential across a range of pathological conditions.

Figure 2. The RNA interference process. siRNA, derived from dsRNA by Dicer, guides RISC to cleave the target mRNA through full complementary binding. (Miao et al., 2024)

Figure 3. miRNA regulates gene expression by guiding the RISC complex to partially complementary mRNA targets, leading to their repression, degradation, or cleavage. (Miao et al., 2024)

Aptamers

Aptamers are synthetic short single-stranded DNA or RNA sequences that bind to various target molecules with high affinity and specificity. By binding to specific target molecules through their three-dimensional structures, aptamers can interfere with the functions of their targets. They can act as inhibitors or agonists, regulating the biological activity of target molecules, such as blocking protein-protein interactions or activating receptor signaling pathways. Aptamers can also serve as targeting moieties, delivering therapeutic agents or imaging probes specifically to diseased tissues, enhancing therapeutic efficacy, and reducing side effects.

Figure 4. Leveraging precision medicine and aptamers for targeted disease therapies. (Zhu et al., 2018)

Small Activating RNA (saRNA)

saRNA is a type of double-stranded RNA that upregulates gene expression. saRNAs are incorporated into RISC, forming an endogenous transcription complex that guides the complex to promoter or enhancer regions of target genes, enhancing transcription factor binding and gene transcription levels.

Single-guide RNA (sgRNA)

sgRNA is a component of the CRISPR gene-editing tool. It guides the Cas9 nuclease to specific genomic loci. The sgRNA-Cas9 complex directs Cas9 to the target DNA site, inducing double-strand breaks, which trigger cellular repair mechanisms to achieve gene insertion, deletion, or replacement.

Transfer RNA (tRNA)

tRNA is a single-stranded RNA, typically 73-93 nucleotides long, primarily involved in protein translation. Recent studies have shown that tRNA can generate small non-coding RNAs under stress conditions, participating in cell proliferation and tumor progression. Under stress conditions, tRNA is specifically cleaved to generate tRNA-derived fragments (tRFs), which regulate gene expression, the cell cycle, and stress responses, potentially playing roles in diseases such as cancer.

Advantages and Challenges of Small Nucleic Acid Drugs

Small nucleic acid drugs offer significant advantages, primarily through their precise regulation of gene expression. Unlike traditional small molecule drugs that alter enzyme activity or receptor binding, small nucleic acid drugs directly target specific RNA molecules, enabling efficient and specific regulation of gene expression. This unique mechanism makes them promising for treating genetic diseases, cancer, and certain infections. For instance, ASO and siRNA have been successfully used in clinical settings to treat genetic disorders like Spinal Muscular Atrophy (SMA) and Duchenne Muscular Dystrophy (DMD).

However, the development of small nucleic acid drugs faces several challenges:

1. Drug Stability: Small nucleic acid drugs are prone to degradation by nucleases in the body, leading to poor stability. To address this, various chemical modification techniques, such as 2'-O-methylation and 2'-fluoro modifications, have been developed to enhance drug stability and improve bioavailability.

2. Delivery Issues: Effective delivery remains a key challenge in the development of small nucleic acid drugs. Due to their unique molecular structure, these drugs require specialized delivery systems to overcome biological barriers and reach target tissues. Current delivery systems include lipid nanoparticles, polymer nanoparticles, and GalNAc conjugates, but ongoing optimization is needed to improve delivery efficiency and targeting.

3. Off-Target Effects: Off-target effects refer to unintended interactions of small nucleic acid drugs with non-target genes, which can reduce specificity and potentially cause side effects. Researchers are working to design more selective molecules and develop high-throughput screening technologies to assess drug targeting.

4. Immune Response: Small nucleic acid drugs may trigger immune responses, affecting their safety and long-term efficacy. The immune system might recognize these exogenous nucleic acids as foreign, leading to immune attacks. Strategies to evade immune detection and tolerance modulation are being explored to address this challenge.

Considerations in Preclinical Drug Development

Preclinical drug development for small nucleic acid drugs involves several critical considerations:

1. Chemical Modifications: Chemical modifications are crucial for enhancing drug stability and reducing immune stimulation. Common modifications include 2'-O-methylation, 2'-fluoro modifications, and phosphate backbone alterations. These modifications improve drug stability, and pharmacokinetic properties, and reduce degradation.

2. Drug Delivery: Effective delivery systems are essential for the development of small nucleic acid drugs. Lipid nanoparticles and GalNAc conjugates are two major delivery systems. Lipid nanoparticles enhance drug stability in the body, while GalNAc conjugates provide targeted delivery to liver-specific cells. Optimizing these delivery systems is vital for improving bioavailability and targeting.

3. Efficacy Validation Models: Emerging technologies such as organoids, organ-on-a-chip systems, and 3D cell culture systems, as well as traditional animal models, are important for evaluating drug efficacy. These models provide in-depth data on drug effectiveness and help predict clinical performance.

4. Pharmacokinetics (PK): Comprehensive evaluation of drug absorption, distribution, metabolism, and excretion is necessary. The choice of administration route (e.g., intravenous, subcutaneous, or oral) significantly impacts pharmacokinetic properties. PK studies reveal drug behavior in the body and assist in optimizing dosing regimens.

5. Safety Evaluation: Assessing off-target effects and immunotoxicity is crucial for ensuring drug safety. Long-term toxicity, especially after extended use, needs careful monitoring to confirm drug safety and tolerability.

Regulatory and Policy Considerations in Drug Development

Following regulatory guidelines and principles is essential during drug development. International guidelines from organizations such as the International Council for Harmonisation (ICH) and the Oligonucleotide Safety Working Group (OSWG) provide detailed requirements for pharmacokinetics, safety pharmacology, and drug metabolism and pharmacokinetics (DMPK) studies. Key considerations include:

1. On-Target and Off-Target Toxicity: Evaluating the toxicity of drugs to both target and non-target genes to ensure specificity and safety.

2. Safety Pharmacology: Assessing the impact of drugs on cardiovascular and central nervous systems to confirm safety across major physiological systems.

3. DMPK Studies: Evaluating drug distribution, metabolism, excretion, and drug interactions to understand drug behavior and minimize adverse drug interactions.

Market and Development Status

Since 1978, the development of small nucleic acid drugs has progressed through exploratory, turbulent, and rapid development phases. Initially, research focused on well-understood viral infections like HIV, cytomegalovirus, and hepatitis C virus, with only cytomegalovirus infection treatments achieving success. Early attempts to develop drugs for common diseases and cancers, such as lung cancer and melanoma, failed due to immature delivery technologies, leading to clinical setbacks. Consequently, many large pharmaceutical companies abandoned small nucleic acid development platforms, and capital withdrew from the field, resulting in a downturn in development.

A few companies, such as Alnylam and Ionis, continued their research, shifting focus to rare genetic disorders with clearer pathophysiological mechanisms. Policies favoring rare diseases also facilitated quicker clinical validation and market entry while avoiding intense competition with drugs for common diseases. Advances in stabilization and targeted delivery technologies revived the field. Between 2013 and 2018, six small nucleic acid drugs for rare diseases were approved. Subsequently, the field rapidly developed, with seven new drugs launched in the past four years. These drugs have shorter clinical trial periods of 5-6 years compared to traditional drug development.

As of 2022, the global market for small nucleic acid drugs was approximately $3.784 billion, showing double-digit growth in recent years. This growth reflects the broad recognition and application of small nucleic acid drugs in medicine. Despite the first small nucleic acid drug, Fomivirsen, being introduced in 1998, the market continues to expand rapidly, growing from $2 billion in 2018 to nearly $4 billion in 2022. 19 small nucleic acid drugs have been approved, including four newly approved drugs: siRNA drug Rivfloza, antisense oligonucleotide drugs Qalsody and Wainua, and aptamer drug Izervay. These drugs target rare diseases such as Duchenne Muscular Dystrophy (DMD), rare lipid disorders, Spinal Muscular Atrophy (SMA), and Amyotrophic Lateral Sclerosis (ALS). With ongoing research and technological advancements, small nucleic acid drugs are expected to play a significant role in the treatment of more diseases in the future.

Creative Biogene: Pioneering Solutions for Small Nucleic Acid Drug Development

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