The target spectrum and treatable diseases of small nucleic acid drugs are diverse. Relevant companies primarily focus on rare monogenic diseases with clear pathogenic mechanisms, well-defined biomarkers, and a lack of effective therapies in the early stages of industry development. They adopt a low-risk strategy to advance early clinical pipelines, seeking rapid target validation and regulatory approval. After the maturity of chemical modification and GalNAc delivery systems, they gradually transition to common diseases related to the liver, cardiovascular system, and metabolism (such as high cholesterol, hepatitis B, diabetes, NASH), and explore targeting extrahepatic tissues/organs, establishing long-term strategies in areas like central nervous system and ophthalmology.
Advantages of Small Nucleic Acid Drugs
Compared to existing therapies, small nucleic acid drugs offer significant advantages. They have a faster target screening process, higher success rates in development, lower likelihood of resistance development, broader therapeutic areas, and longer duration of action, making them highly promising. Currently, small nucleic acid drugs have shown preliminary evidence in clinical settings for curing diseases, replacing existing therapies, and addressing unmet medical needs, with breakthrough products like Leqvio and Spinraza. With continuous technological advancements, small nucleic acid drugs are poised to become a disruptive new mainstream therapy following small molecules and antibodies.
a. Shorter development cycles and rapid target screening. Unlike small molecules and antibody drugs, which require identifying complex spatial conformations of proteins and thus extensive drug screening, small nucleic acid drugs only need to target disease-causing gene sequences and design and synthesize corresponding RNA fragments, leading to much faster early-stage development.
b. Lower likelihood of resistance development. While antibodies and small molecules mainly exert therapeutic effects by regulating cellular signaling pathways and metabolism, which may lead to resistance due to compensatory pathway upregulation or decreased antigen expression, small nucleic acid drugs directly regulate upstream gene expression, making them relatively resistant-resistant.
c. Broader therapeutic areas. Not limited by protein druggability, small nucleic acid drugs can theoretically be designed to target any gene of interest, requiring only sequence information of the target mRNA, potentially tackling genetic diseases and other hard-to-treat diseases.
d. Safety. Small nucleic acid drugs have low dosages and frequencies, mimic natural biological processes, can be biodegraded, have low cytotoxicity and immunogenicity, intervene at the mRNA level, and have no impact on the genome.
e. Long-lasting effects. Typically, the half-life of small molecule drugs is measured in hours, and that of antibody drugs is measured in days/weeks, whereas small nucleic acid drugs can be circulated and reused multiple times in the body, reducing dosing frequency, and their half-life in the body can be calculated in months, providing significant clinical value for many diseases, especially chronic ones.
f. Higher clinical development success rates. Due to the clear mechanism of action, small nucleic acid drugs function by completing Watson-Crick base pairing with mRNA to achieve their function, without requiring complex protein structures. Hence, they have a relatively high success rate in development. Referring to the development success rate of Alnylam, the success rates from Phase I to Phase III reach 59.2%, which is 5 times higher than that of targeted drugs and the overall pharmaceutical industry.
Challenges and Solutions for Small Nucleic Acid Drugs
Currently, nucleic acid-based therapies face several challenges, including off-target effects, efficacy, half-life, delivery, immune response, and toxicity. Despite many oligonucleotide-based drugs relying on sequence-specific interactions and/or triggering specific enzyme activity to bind to their target mRNA, off-target effects persist and are concentration-dependent. Enhancing the stability of nucleic acid drugs against nucleases through different chemical modifications or effective delivery systems may compromise their efficacy. Optimization of nucleic acid drugs and their delivery systems through optimal chemical modifications is essential.
In addition to the challenges of nucleic acid drugs themselves, nucleic acid drugs as polyanionic biomolecules cannot diffuse across cell membranes due to their size and negative charge. For instance, in the case of siRNA, clinical siRNA delivery faces several obstacles, including intravascular degradation, extracellular and tissue-specificity, intracellular transport, and extracellular matrix. Unmodified nucleic acids have a short half-life in circulation due to susceptibility to intravascular degradation and rapid renal clearance. Many traditional chemical modifications of nucleic acids, especially oligonucleotides, have been achieved, such as PS linkages, 2′-OMe, 2′-MOE, 2′-F, and LNA. These chemically modified nucleic acid drugs can enhance their pharmacokinetic properties in vivo, greatly increase resistance to nucleases, enhance binding affinity, and reduce immune responses.
Nucleic acid nanoparticles have the potential to address challenges in nucleic acid-based therapies. Nanoparticle-based gene delivery can improve the half-life of nucleic acids in circulation and protect nucleic acid drugs from intravascular degradation. Moreover, the material, shape, and surface functionalization of nanoparticles greatly influence nucleic acid packaging and in vivo systemic delivery.
Specific tissue accumulation, cellular internalization, and nucleic acid endosomal escape are common challenges in in vivo gene-based therapy. Therefore, effective delivery systems for nucleic acid drugs must include the following features: (1) enhanced stability of nucleic acid drugs against various nucleases, (2) non-stimulation of the innate immune system, (3) prevention of renal clearance, (4) targeting specific cells from bloodstream transport to specific tissues, (5) high payload efficiency and efficient endosomal escape.
DNA/RNA Modification at BOC Sciences
Chemical Modifications of Small Nucleic Acid Drugs
The chemical modifications of nucleic acid drugs aim to improve their specificity for target delivery in vivo, enhancing both targeting and delivery properties. There are several ways nucleic acid drugs can be chemically modified, including modifications of the backbone, bases, sugars, and incorporation of non-natural nucleotides.
Cholesterol-modified siRNA
Conjugation of cholesterol with siRNA (Chol-siRNA) increases the net lipophilicity of Chol-siRNA conjugates compared to naked siRNA, improving their cellular uptake and silencing efficiency. After systemic administration, chemically stable Chol-siRNA typically associates with lipoproteins (such as HDL, LDL) in the bloodstream to form lipoprotein particles. Studies have shown that after intravenous injection and cholesterol modification, the pharmacokinetic properties of Chol-siRNA are significantly improved (e.g., half-life, clearance rate), and cholesterol modification also enhances broad tissue biodistribution.
Alkyl chain-modified siRNA
Lipophilic siRNA comprises therapeutically active siRNA modified with hydrophobic lipids (e.g., bile acids) and long-chain fatty acids. Cellular uptake and therapeutic gene silencing activity of lipophilic siRNA depend mainly on the length of the alkyl chain between siRNA and lipophilic residues. It has been reported that the efficacy or affinity of lipophilic siRNA binding to lipoprotein particles correlates with cellular uptake and therapeutic silencing activity in vivo. The optimal uptake and silencing activity are observed with Chol-Cx-siRNA containing alkyl linkers with 6 to 10 carbon atoms. Longer alkyl linkers improve cellular uptake of siRNA but reduce its silencing efficiency on target genes.
Vitamin E-modified small nucleic acids
Vitamin E (VitE, tocopherol) is a low-toxicity, essential, naturally occurring lipophilic antioxidant that cannot be synthesized in human cells. VitE modification has been shown to improve gene delivery of nucleic acid drugs in vitro and in vivo, similar to cholesterol modification. Amphiphilic VitE-modified oligonucleotides have larger surface areas due to the hydrophobic VitE head and hydrophilic oligonucleotide tail. VitE-modified oligonucleotides can interact with cell surface receptor proteins for cell-type-specific capture and delivery. Hydrophobic tocopherols on modified ASOs can bind to intracellular organelles or intracellular proteins and assist ASOs in entering cells. However, the binding of tocopherol to binding proteins greatly inhibits the acquisition of antisense oligonucleotides for targeting mRNA and/or the interaction of RNase H and ASO/mRNA duplexes, resulting in loss of ASO gene knockdown. However, Toc-ASOs with 4-7 degradable linkers significantly increase systemic gene delivery and therapeutic silencing activity in vivo.
N-acetylgalactosamine (GalNAc)-modified oligonucleotide
GalNAc is a well-known ligand for liver targeting, with high affinity for asialoglycoprotein receptors (ASGPR). Single, double, triple, or quadruple GalNAc-modified oligonucleotides can improve the delivery efficiency of oligonucleotide drugs to liver cells. GalNAc-modified oligonucleotides can be specifically delivered to liver cells via ASGPR-mediated endocytosis, significantly enhancing the inhibition of target gene expression in liver cells. GalNAc-conjugated siRNA/ASO drugs administered by subcutaneous injection (intravenous administration is rapidly cleared by the kidneys) extend the circulation time, allowing for dosing once every few months to half a year.
Dynamic covalent polymer (DCP) oligonucleotides
The first-generation Dynamic Covalent Polymer (DPC 1.0) system consists of reversible, amphiphilic, and water-soluble polymers (PBAVE polymers) with amino and butyl side chains modified with masking agents (such as PEG reagents and GalNAc ligands) through acid-labile carboxylic acid dimethylmaleic anhydride (CDM) linkages. The second-generation DCP (DCP 2.0) has an amide-like peptide (MLP) scaffold connected to masking agents via CDM linkages. DCP’s CDM linkages are easily degraded in the acidic environment of endosomes, leading to the release of masking agents and protonation of the scaffold. DPCs, as efficient and specific non-viral vectors, are widely used for in vivo siRNA delivery to liver cells, with the advantage of releasing siRNA from endosomes.
Nucleic Acid aptamer-oligonucleotide conjugates
Cell-type-specific aptamers, often referred to as “chemical antibodies,” have advantages such as easy chemical solid-phase synthesis, physical and thermal stability, and low or no immunogenicity. In addition to being developed as nucleic acid drugs themselves, nucleic acid aptamers can also be conjugated with other nucleic acid drugs for targeted delivery. Aptamer-siRNA/shRNA/miRNA hybrids consist of an aptamer, a therapeutic oligonucleotide (siRNA, miRNA, or shRNA) linked via an alkyl portion, and biotin-streptavidin interaction, complementary oligomer hybridization, or sticky bridge. Aptamer-siRNA hybrids targeting prostate-specific membrane antigen (PSMA) are the first generation of chimeric RNA aptamers targeting PSMA-expressing cells and inhibit PLK1 and BCL2 gene expression. Two biotinylated PSMA aptamers and two biotinylated siRNAs independently bind to four binding sites of streptavidin, and the aptamer-siRNA complex is specifically delivered to PSMA-expressing cells, leading to effective gene silencing of prostate-specific membrane antigen A/C.
Peptide or antibody-modified oligonucleotides
To achieve targeted delivery of nucleic acid drugs to specific cells or tissues, researchers also use different targeting peptides or antibodies coupled with nucleic acid drugs, among which cyclic RGD (cRGD) peptide is one of the most relevant and commonly used peptides in gene delivery applications. cRGD maintains high specificity and affinity for αvβ3 integrin, which is often overexpressed on the surface of many cancer cells (such as melanoma, brain tumors, and breast cancer). In addition, antibodies are also used, where conjugation of nucleic acid drugs with a certain antibody enables the targeted delivery of nucleic acid drugs to specific cells or tissues.
Delivery Systems
Lipid Nanoparticles (LNPs)
siRNA encapsulated in lipid nanoparticles is typically more stable in the bloodstream than naked siRNA, which enhances the safety, tolerability, and pharmacokinetics of siRNA. The surface of lipid nanoparticles can further be functionalized with PEG reagents and various targeting ligands. Additionally, the composition of lipid nanoparticles can be modified with other lipids through post-insertion methods. These functionalizations of lipid nanoparticles address key challenges in gene delivery, such as prolonged circulation in the bloodstream, specific cellular uptake in target tissues, and efficient endosomal escape. There are four types of lipid nanoparticle structures for nucleic acid drug delivery, including lipid complexes, stable nucleic acid lipid particles (SNALPs), lipid multi-complexes, and membrane-core nanoparticles (MCNPs). SNALPs are considered promising for systemic delivery of nucleic acid drugs, especially siRNA drugs, in clinical applications.
Peptide nanoparticles (PNPs)
Composed of histidine/lysine polymers (HKPs), PNPs aggregate into nanoparticles, encapsulating and protecting 10k~100k siRNA for delivery to target tissues and cells via the NRP1 receptor. After entering cells, protonation mediated by histidine enhances the endosomal escape efficiency, releasing the siRNA payload to the cytoplasmic action site.
Polymeric nanoparticles
siRNA conjugated with neutral hydrophilic PEG polymers (PEG-siRNA conjugates) has been shown to be useful for systemic gene delivery.
Metallic nanoparticles
Metallic nanoparticles are also used as gene delivery vectors due to their low toxicity, good biocompatibility, high delivery efficiency, and ease of surface functionalization. Oligonucleotides can attach to the surface of metallic nanoparticles through chemical modification and electrostatic interactions. Functionalization methods of metallic nanoparticles with oligonucleotides include salt aging, pH reduction, and freezing. Gold nanoparticles (Au NPs) have been shown to efficiently conjugate with DNA modified with terminal thiols after dry ice freezing for 2 minutes.
Charge-reversed nanoparticles
Charge-reversed nanoparticles are formed by a group of positively charged polymers sensitive to intracellular species. Charge reversal of the polymer from positive to negative results in the release of condensed therapeutic oligonucleotides. Charge-reversed nanoparticles have been demonstrated to be effective gene delivery agents both in vitro and in vivo.
Self-assembled oligonucleotide nanoparticles (ONPs) with three-dimensional structures have potential biological applications in imaging and delivery in vivo. It has been reported that DNA cages formed by self-assembled synthetic DNA oligonucleotides are nano-scale tetrahedral structures used for delivery and release of cargo in human embryonic kidney (HEK) cells.
RNA transcript nanoparticles
RNAi microsponges are a novel siRNA carrier containing numerous polymerized hairpin RNA fragments that self-assemble into nucleic acid complexes. In the presence of positively charged PEI, RNAi microsponges can further condense into dense nanoparticles for efficient delivery of siRNA in vitro or in vivo. Upon cellular uptake, the hairpin RNA in RNAi microsponges is converted into siRNA by Dicer in the RNAi pathway.
People also like
Lipid Nanoparticles (LNPs) Design and Formulation Production
Overview of Lipid Nanoparticle
siRNA vs ASO: Small Nucleic Acid Drugs Revolution Unleashed
Marketed Nucleic Acid Drugs and Their Lipid Nano-Delivery Systems
FDA-Approved Nucleic Acid Drugs
References
- Chen C, Yang Z, Tang X. Chemical modifications of nucleic acid drugs and their delivery systems for gene-based therapy. Med Res Rev. 2018;38(3):829-869. doi:10.1002/med.21479
- Ramasamy T, Ruttala HB, Munusamy S, Chakraborty N, Kim JO. Nano drug delivery systems for antisense oligonucleotides (ASO) therapeutics [published correction appears in J Control Release. 2023 Feb;354:34]. J Control Release. 2022;352:861-878. doi:10.1016/j.jconrel.2022.10.050
- He F, Wen N, Xiao D, et al. Aptamer-Based Targeted Drug Delivery Systems: Current Potential and Challenges. Curr Med Chem. 2020;27(13):2189-2219. doi:10.2174/0929867325666181008142831
- Eygeris Y, Gupta M, Kim J, Sahay G. Chemistry of Lipid Nanoparticles for RNA Delivery. Acc Chem Res. 2022;55(1):2-12. doi:10.1021/acs.accounts.1c00544