In recent years, nucleic acid drugs have become a research hotspot in the treatment of various incurable diseases due to their advantages such as simple design, short research and development cycle, high success rate, strong specificity, and the ability to fundamentally regulate pathogenic genes. However, nucleic acid drugs also have some issues, including poor stability in vivo, susceptibility to degradation by nucleases or clearance by the kidneys; their large molecular size and negative charge make it difficult for them to cross cell membranes to function inside cells; weak targeting ability in vivo, which may fail to accurately target target cells or tissues, potentially leading to off-target effects, causing side effects and damaging the body; and once inside cells, they may face the risk of being hydrolyzed by lysosomes or non-specifically adsorbed by proteins during the circulatory process, rendering them ineffective. Therefore, in addition to improving the stability and immunogenicity of nucleic acids through chemical modifications, efficient and safe delivery systems in vivo are also needed. Currently, the development of nucleic acid drugs can be divided into two major directions: chemical modification and physical modification, which can individually or jointly enhance the stability of nucleic acid drugs both inside and outside the body and the translation efficiency within specific tissues and cells. Various nucleic acid modification and delivery techniques have been developed, mainly including chemical modification, ligand conjugation, and nano-delivery carriers.

Chemical Modification

Chemical modification of nucleic acids is widely used in nucleic acid drugs such as ASO, siRNA, miRNA, and mRNA, which helps these drugs play a role in disease treatment. Modifying nucleic acid drugs can enhance their resistance to enzymatic degradation, maintain sequence stability, and prolong the half-life, as well as increase their lipophilicity and reduce immunogenicity. Furthermore, chemical modification can improve targeting ability, such as introducing ligands or aptamers that specifically recognize cell surfaces at the ends of nucleic acid sequences, thereby enhancing the silencing efficiency and catalytic reaction efficiency of nucleic acid drugs.

1、Phosphate Group Modification

Phosphate group modification often occurs on the non-bridging oxygen atoms, and a widely used method is the substitution of a sulfur atom for one of the non-bridging oxygen atoms of the phosphate group, forming a phosphorothioate bond. This phosphorothioate bond helps to enhance the resistance of nucleic acids to enzymatic degradation and their ability to bind to plasma proteins, thus prolonging their circulation time in the body. However, the disadvantage of sulfur modification is its weaker binding affinity to target sequences, and high levels of phosphorothioate bonds may lead to cellular toxicity and immune stimulation side effects. Therefore, the position and number of phosphorothioate bonds are also important for delivery efficiency. In addition, the oxygen atoms on the phosphate group can also be substituted by various amine groups, boronic alkyl groups, or the entire phosphate group can be replaced by amide groups, aminooxy groups, alkoxy groups, triazolyl groups, etc., but these applications are not as common as sulfur modification.

2、Base Modification

The modification of bases primarily involves modifying substituent groups on the bases or replacing them entirely. The 5-position of pyrimidines and the 8-position of purines are commonly used substitution sites. Common types of base modifications include pseudouridine, 2-thiouridine, N1-methylpseudouridine, 5-methyluridine, 5-methoxyuridine, N6-methyladenosine, and 5-methylcytidine. Among them, replacing uracil with pseudouridine is one of the common base modifications. Research has shown that replacing uracil with pseudouridine can improve the translation of mRNA encoding Cas9 and reduce its immunogenicity. In addition, N-ethylpiperazine-6-triazole-modified adenosine analogs can block the interaction between siRNA and Toll-like receptor 8, reducing immunogenicity. 6'-Phenyl-pyrrolocytosine is a cytosine analog that exhibits good base-pairing effects, thermal stability, and strong fluorescence. siRNA modified with 6'-phenyl-pyrrolocytosine maintains its gene silencing effect and its fluorescent properties can be used to detect the uptake and transport of siRNA within cells.

3、Ribose Modification

Ribose modification primarily includes two types: introducing different sizes and polarities of groups at the 2' position, such as 2'-methoxy, 2'-methoxyethoxy, and 2'-deoxy-2'-fluoro; and modifying the 2' position and other ribose sites simultaneously, such as locked nucleic acid (LNA), peptide nucleic acid (PNA), and phosphoramidite morpholino oligonucleotides (PMO). The 2' modification is crucial for inhibiting the hydrolysis of nucleases. 2'-Methoxy modification is a widely used ribose modification method, which can enhance the binding affinity of drugs to target mRNA, inhibit the hydrolysis of nucleases, reduce immunogenicity in vivo, and impart a certain degree of lipophilicity to the nucleic acid structure. 2'-Methoxyethoxy is an analog of 2'-methoxy and has similar properties, but it exhibits stronger affinity and anti-enzymatic degradation ability for target mRNA. 2'-Deoxy-2'-fluoro modification can improve the affinity and stability of siRNA. Modifying the 2', 4' positions or even the entire sugar ring of ribose can also produce good results.

Locked nucleic acid (LNA) is a restrictive modification of nucleic acid conformation, adopting a C3'-endo conformation. It can maintain high target affinity and anti-enzymatic degradation ability with shorter sequences. However, the off-target effects and toxicity issues of short-sequence LNA are more severe. Therefore, in practical applications, unlocked nucleic acid, constrained ethyl-bridged nucleic acid, tricyclic DNA, and glycol nucleic acid are often used as alternatives. Combining these four ribose modifications with phosphorothioate modification can achieve better therapeutic effects. The electrically neutral structure brought by PMO and peptide nucleic acid modifications can improve the stability and affinity of nucleic acid drugs. PMO has good water solubility and is widely used. Currently, some PMO-modified siRNAs and miRNAs have entered clinical trial stages. PMO can also be further modified, such as by introducing cell-penetrating peptides and positively charged amine groups, to improve its transmembrane capability.

Conjugated Ligands

Nucleic acid drugs can prolong their circulation time in the body, increase accumulation and uptake in specific tissues and cells by covalently conjugating with lipids, galactose, proteins/peptides, or aptamers. Utilizing specific conjugated ligands not only modulates the binding of nucleic acid drugs to plasma proteins, improving drug tissue distribution, but also achieves targeted delivery to specific tissues or cells through ligand targeting of cell surface receptors. Additionally, by designing the linker between the ligand and the nucleic acid drug, such as using acid-sensitive amide bonds, disulfide bonds that are easily reduced and broken in the cytoplasm, or Dicer-sensitive bonds, the nucleic acid drug can be separated from the conjugate after entering a specific physiological environment, effectively adapting to specific therapeutic mechanisms.

1、Lipid Conjugation

Conjugates formed by the covalent binding of oligonucleotides to lipids and their derivatives can enhance the in vivo delivery efficiency of oligonucleotides. For example, siRNA covalently bonded with cholesterol at the 3' end can effectively silence myostatin to induce muscle growth, and siRNA covalently bonded with α-tocopherol can silence ApoB in mouse livers. RNA conjugated with cholesterol can increase the uptake by specific tissues and cells and alter the distribution of siRNA through pre-assembly with high-density lipoprotein (HDL) or low-density lipoprotein (LDL). Compared to free siRNA-cholesterol, siRNA-cholesterol pre-assembled with HDL (HDL siRNA) can produce a stronger silencing effect in the liver and jejunum. Further research has found that siRNA-cholesterol particles pre-assembled with LDL (LDL siRNA) are almost exclusively absorbed in the liver, while HDL siRNA particles are primarily absorbed by the liver, adrenal glands, ovaries, kidneys, and small intestine. Therefore, researchers have proposed that the endocytosis of siRNA-cholesterol is mediated by HDL- and LDL-related scavenger receptor class B type I and LDL receptors, respectively. Other studies have found that the in vivo binding efficacy of siRNA-lipid conjugates to different lipoprotein classes is influenced by their hydrophobicity, with more hydrophobic conjugates preferentially binding to LDL and less hydrophobic conjugates preferentially binding to HDL. Therefore, targeted delivery of RNA drugs to different tissues can be achieved by conjugating lipids with different hydrophobicity.

2、N-Acetylgalactosamine (GalNAc) Conjugation

GalNAc is a disaccharide compound that has a high affinity for the asialoglycoprotein receptor (ASGPR). ASGPR is highly specifically expressed in the liver, and GalNAc-conjugated RNA drugs are expected to achieve efficient and specific delivery to the liver. In 2019, the first GalNAc-conjugated siRNA drug, Givosiran, targeting delta-aminolevulinic acid synthase 1, was approved for the treatment of patients with acute intermittent porphyria. By the end of 2020, two GalNAc-conjugated siRNA drugs, lumasiran and inclisiran, were also approved for clinical use. Lumasiran is an siRNA drug used to treat primary hyperoxaluria type 1, reducing the translation of glycolate oxidase 1 mRNA in hepatocytes. Inclisiran is an siRNA drug used to treat atherosclerotic cardiovascular disease, reducing the translation of proprotein convertase subtilisin/kexin type 9 mRNA. However, ASGPR is only highly expressed in highly differentiated hepatocytes and is expressed at lower levels in poorly differentiated hepatoma cell lines, so the strategy of using GalNAc conjugation for the treatment of liver cancer still needs to be improved. Additionally, due to the lack of ASGPR expression in extrahepatic tissues, efficient RNA drug delivery using GalNAc conjugation technology has not yet been achieved.

3、Peptide Conjugation

Cell-penetrating peptides (CPP) are typically amphiphilic or cationic peptide fragments composed of 5 to 30 amino acids, including HIV-1 (human immunodeficiency virus-1) transactivator of transcription (HIV-Tat), the third fragment of the Drosophila antennapedia homeodomain DNA-binding domain (penetratin 1), chimeric peptides synthesized from neuropeptide galanin and lactoferrin (transportan), or polymers based on basic amino acids (such as arginine and lysine). When CPPs are conjugated with negatively charged nucleic acids or electrically neutral modified nucleic acids, they can increase the overall positive charge of the conjugate, promoting cellular uptake of the nucleic acid. Currently, several peptide-PMO (peptide-phosphorodiamidate morpholino oligomer, PPMO) conjugates are in preclinical research stages. PPMO M23D-B can enhance the skipping of dystrophin exon 23, achieving sustained expression of dystrophin in the muscles of disease model mice. The arginine-rich Pip6a peptide conjugated with PMO (Pip6a-PMO) can directly carry nucleic acids into the central nervous system of spinal muscular atrophy model animals, enhancing the expression of survival motor neuron protein by targeting CUG repeat-expanded transcripts, treating myotonic dystrophy type 1 (DM1). Besides CPPs, GLP1-like peptides targeting the glucagon-like peptide 1 receptor (GLP1R) and multivalent cyclic arginylglycylaspartic acid (cRGD) peptides have also been shown to mediate RNA delivery. Researchers modified ASO with a 40-amino acid GLP1-like peptide, enabling silencing of target genes in pancreatic β-cells. cRGD-modified siRNA can specifically deliver to melanoma cells overexpressing αvβ3 integrin, knocking down target genes. These studies suggest that peptide conjugation can improve the tissue distribution and cellular uptake of RNA. However, there is also a possibility that the attraction between peptides and RNA due to opposite charges can lead to aggregation, affecting the delivery efficiency of RNA.

4、Antibody/Aptamer Conjugation

Currently, three non-fusogenic PMO drugs, eteplirsen, golodirsen, and viltolarsen, have been approved for the treatment of Duchenne muscular dystrophy, yet their effects on muscle improvement remain to be improved. Researchers have attempted to enhance oligonucleotide drug delivery for muscle diseases through receptor-mediated methods, such as conjugating antibodies to the transferrin receptor (TfR) to increase muscle tissue delivery efficiency. TfR, as the main entry for iron-bound transferrin, is highly expressed in skeletal muscle cells, cardiomyocytes, brain endothelial cells, and proliferative tumor cells with high metabolic activity, and can internalize conjugates through clathrin-mediated endocytosis. Through this pathway, TfR can facilitate the entry of siRNA-antibody/aptamer conjugates into muscle tissue or across the blood-brain barrier into the central nervous system, silencing the expression of specific genes. Based on receptor-ligand interactions, multiple receptors have been proven to enhance the targeted delivery efficiency of siRNA and ASO, including human epidermal growth factor receptor 2, T-cell marker CD7, transferrin receptor CD71, CD44, laminin receptor-2, and epidermal growth factor receptor.

Nucleic acid aptamers are single-stranded DNA or RNA molecules with a length of 20 to 100 nucleotides that can form specific and stable tertiary structures, enabling them to bind to target molecules with high specificity and stability. They are therefore also known as "chemical antibodies." Compared to protein antibodies, nucleic acid aptamers are easier to manufacture, cost less, are smaller in size, and have lower immunogenicity. By conjugating nucleic acid aptamers with specific RNA drugs, the targeted delivery of the target RNA in vivo can be enhanced.

The prostate-specific membrane antigen (PSMA) aptamer A10 is the first-generation nucleic acid aptamer that targets PSMA-positive cells. siRNA conjugated with A10 can significantly inhibit the expression of PLK1 and BCL2 genes in PSMA-positive cells, achieving silencing of the lamin A/C gene. Similarly, the RNA aptamer of gp120 can be linked to siRNA through a short nucleic acid sequence rich in GC modified with 2'-OMe and 2'-fluoropyrimidine to form a complex that inhibits HIV-1 replication in model mice.

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References

[1] Li Dan, Huang Yukun, Gao Xiaoling. Research Progress in RNA Drug Delivery [J]. Acta Pharmaceutica Sinica, 2023, 58(03)

[2] Wang Junfeng, Tan Manman, Wang Ying, et al. Research Progress in the Modification and Delivery of Nucleic Acid Drugs [J]. Journal of Zhejiang University (Medical Sciences), 2023, 52(04)

About the Author

Xiaomichong, a pharmaceutical quality researcher, has been committed to pharmaceutical quality research and drug analysis method validation for a long time. Currently employed by a large domestic pharmaceutical research and development company, she is engaged in drug inspection and analysis as well as method validation.