The ability of small nucleic acids to regulate gene expression through a series of processes has been extensively explored. Compared to traditional therapies, small nucleic acid therapeutics hold the potential to achieve durable or even curative effects through gene editing. In recent years, due to technological advancements, efficient delivery of small nucleic acids for therapeutic and biomedical applications has been achieved, accelerating the clinical translation of small nucleic acid-based systems.

Background of Small Nucleic Acid Therapeutics

Gene therapy was initially explored for treating diseases in the 1960s and early 1970s. Significant biological and technological breakthroughs have led to the development of several safe and effective platforms. Nucleic acid therapeutics use engineered nucleotide sequences to selectively modulate gene expression. The most prominent nucleic acid drugs are based on antisense oligonucleotides (ASOs), aptamers, short interfering RNAs (siRNAs), and microRNAs (miRNAs). These small nucleic acids have revolutionized precision medicine and enhanced the efficacy of existing drugs by improving the action and delivery of therapeutic agents. Oligonucleotides can be designed to target specific mRNAs and are capable of treating or managing a wide range of diseases. The mechanisms of action of these drugs differ from traditional drugs, which in some cases helps prevent drug resistance, particularly in cancer therapy. The efficacy of these drugs can be precisely controlled by altering their sequence, structure, or chemical modifications. Their flexibility allows for customization of the drug according to the specific needs of the disease. In recent years, researchers have developed various delivery platforms to enhance the stability and bioavailability of oligonucleotide drugs, thereby improving their therapeutic effects.

Figure 1: Small nucleic acid therapeutics: from bench to clinic and beyond

Several Types of Small Nucleic Acid Therapeutics and Their Mechanisms 

Currently, small nucleic acid therapeutics include the following six types: (1) ASOs are short, synthetic nucleic acid sequences that bind to complementary mRNA molecules (this binding can block the translation process or promote mRNA degradation, thereby reducing target protein expression); (2) Aptamers are short, single-stranded nucleic acids that bind to specific targets with high affinity and specificity. They function by folding into unique three-dimensional structures that enable them to interact effectively with proteins, small molecules, or cells; (3) siRNAs are double-stranded RNA molecules that guide the RNA-induced silencing complex (RISC) to complementary mRNA targets. This interaction leads to mRNA cleavage and degradation, resulting in the silencing of specific genes; (4) miRNAs are endogenous single-stranded RNA molecules that regulate gene expression post-transcriptionally. They bind to complementary sequences in the 3' untranslated regions (UTRs) of target mRNAs, typically leading to translational repression or mRNA degradation, thereby modulating the expression of multiple genes; (5) Piwi-interacting RNA (piRNA) are small single-stranded RNA molecules, approximately 24-30 nucleotides in length. In germline cells, they interact with PIWI proteins to regulate gene expression, silence transposable elements, and maintain genomic stability; (6) saRNAs are synthetic, short RNA molecules designed to upregulate gene expression. They bind to specific promoter regions of target genes, recruiting transcriptional activators or altering chromatin structure to increase transcriptional activity, thereby increasing the production of specific proteins. The mechanisms of action for these six types of small nucleic acid therapeutics are illustrated below:

Figure 2: Mechanisms of action of six types of small nucleic acids (a. ASOs; b. Aptamers; c. siRNA; d. miRNAs; e. Piwi-interacting RNA (piRNA); f. saRNAs)

Common Modifications in Small Nucleic Acid Therapeutics

Common modifications in small nucleic acid therapeutics can be categorized into three classes: ribose modifications, nucleobase modifications, and backbone modifications. Ribose modifications are frequently performed at the 2' position of the ribose sugar (blue box) and on the sugar chain (orange box). For nucleobase modifications, uracil, adenine, and cytosine are common targets (purple box); the ribose can also be completely replaced (red box). Backbone modifications can alter the PO bonds of the backbone, involving non-bridging O atoms (cyan box), 3' bridging O atoms (green box), and the entire PO bond (golden box), to reduce the net anionic charge of the small nucleic acid.

Figure 3: Chemical modifications of small nucleic acids.

Common Delivery Technologies for Small Nucleic Acid Therapeutics 

Common delivery technologies for small nucleic acid therapeutics can be classified into the following three categories: (1) Non-viral delivery vectors: including lipid nanoparticles (LNPs), polymers, peptides, exosomes, conjugates (GalNAc), and inorganic nanoparticles; (2) Endocytosis pathways: primarily macropinocytosis, caveolin-mediated endocytosis, clathrin-mediated endocytosis, and direct membrane fusion; (3) Endosomal escape: following internalization, endocytic vesicles mature into early endosomes and late endosomes, subsequently fusing with lysosomes, which induces lysosomal degradation. Only a small fraction (typically less than 1%) of small nucleic acids escape from endosomes into the cytoplasm.

Figure 4: Technologies for delivering small nucleic acids (a. Schematic representation of common non-viral delivery vectors (6 types); b. Schematic representation of uptake pathways for small nucleic acid drugs into target cells; c. Schematic representation of endosomal escape).

Challenges and Future Directions

Figure 5: Challenges and future directions for small nucleic acid drugs.

Challenges and future directions for small nucleic acid drugs. Various challenges persist in the development of small nucleic acid drugs (red box). First, the development of new drugs is both expensive and time-consuming. Additionally, nanoparticles (NPs) may induce adverse immune effects, such as inflammatory cytokine storms. Furthermore, cost and regulatory hurdles exist. Insufficient organ and cell selectivity reduces the effectiveness of small nucleic acid drugs. Numerous ideas have been proposed to address these obstacles as future directions for small nucleic acid drugs (green box). The first involves the application of artificial intelligence (AI) in drug discovery. The second is enhancing safety, focusing on both the delivery vector and the small nucleic acid sequence. The third is the surface modification of nucleic acid drugs to enable delivery to specific cell types. Finally, screening vectors for more efficient delivery of nucleic acid drugs. 

This article provides an overview of the expanding landscape of small nucleic acid therapeutics, including their types, mechanisms, common chemical modifications, delivery platforms, as well as challenges and future directions. Currently, an increasing number of small nucleic acids are transitioning from research to clinical applications, yielding promising results. However, strategic issues must also be considered, such as important safety considerations, the development of novel vectors, and overcoming barriers to translate academic breakthroughs into clinical practice. The positive results generated by small nucleic acid therapeutics in clinical trials hold the potential to address previously "undruggable" targets, suggesting that these therapies may help guide the development of other clinical candidates.

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References

[1] Liu, M., Wang, Y., Zhang, Y. et al. Landscape of small nucleic acid therapeutics: moving from the bench to the clinic as next-generation medicines. Sig Transduct Target Ther 10, 73 (2025).