With the continuous elucidation of the structure and function of nucleic acid molecules, as well as significant improvements in nucleic acid synthesis techniques and significant cost reductions, nucleic acid research and drug innovation have gradually become a hot topic in precision biomedicine and disease treatment. Nucleic acids have also emerged as a rising star in the pharmaceutical industry due to their unique chemical and biological functions. Through relentless efforts and exploration, nucleic acid drugs have made significant progress in the treatment of diseases such as viruses, high cholesterol, gene expression editing, vision loss, and hepatic vein occlusion in recent years, demonstrating the enormous potential and advantages of the nucleic acid drug field. Nucleic acid drugs are typically composed of natural or modified deoxyribonucleotides or ribonucleotides, including DNA, RNA, antisense oligonucleotides (ASOs), and nucleic acid aptamers. Currently, nucleic acid drugs with faster research progress and clinical applications mainly include ASOs, siRNA, mRNA, and nucleic acid aptamers.

1.ASOs Drugs

Antisense oligonucleotides (ASOs) are a common type of DNA-based drugs, typically consisting of single-stranded short DNA sequences ranging from 13 to 30 base pairs. ASOs utilize oligonucleotide sequences complementary to the target gene, forming a DNA/RNA double-stranded structure at the target site, resulting in the inhibition of gene function. In addition to targeting mRNA genes, ASOs can also target microRNAs (miRNAs), and the ASOs targeting miRNAs are referred to as antimir or antagomir. The mechanism of action of ASOs not only involves forming a strong double-stranded structure that causes steric hindrance, but also inducing RNase H to cleave the RNA strand. RNase H is an endogenous enzyme that recognizes DNA/RNA double strands and specifically cleaves the RNA strand. Therefore, the double strand formed by ASOs can catalyze the targeted cleavage by RNase H.

The research on ASOs began earlier than other nucleic acid drugs. So far, there are 9 antisense nucleic acid drugs approved by the FDA for marketing, and 2 early ASO drugs have been withdrawn from the market due to low sales and other reasons. Currently, there are still 7 on the market.

Vitravene, approved in 1998, was the first nucleic acid drug approved by the FDA for marketing, used to treat cytomegalovirus retinitis in immunocompromised patients (mainly AIDS patients). It specifically binds to the mRNA of cytomegalovirus (CMV) CMV-IE2, and then RNase H recognizes and hydrolyzes the targeted mRNA, ultimately blocking the synthesis of the protein IE2 necessary for CMV replication, thereby inhibiting CMV proliferation and achieving therapeutic effects. Due to the rapid development of highly active antiretroviral therapy (HAART), the number of cytomegalovirus cases has dropped dramatically, and this product has been discontinued.

In January 2013, the FDA approved Kynamro for the treatment of homozygous familial hypercholesterolemia (HoFH). Excessive low-density lipoprotein (LDL) particles are a key factor leading to atherosclerosis, and apolipoprotein B100 (ApoB-100) is the main protein that carries these lipoprotein particles. Therefore, targeting ApoB100 as a drug target is the most important breakthrough in the treatment of HoFH. Kynamro targets ApoB-100 mRNA, specifically binding to ApoB-100 mRNA. RNase H specifically hydrolyzes ApoB-100 mRNA, blocking the synthesis of ApoB-100 and thus reducing cholesterol levels. Currently, this product has also been discontinued.

In September 2016, EXONDYS 51 (eteplirsen) was approved by the FDA for marketing, becoming the first phosphorodiamidate morpholino oligomer (PMO) approved for the treatment of Duchenne muscular dystrophy (DMD). DMD patients suffer from mutations in the DMD gene, resulting in the removal of one or several exons during the process of pre-messenger RNA (Pre-mRNA) forming mRNA, altering the coding reading frame and preventing the expression of functional dystrophin protein due to premature termination codons. Eteplirsen specifically binds to exon 51 of the dystrophin Pre-mRNA, removing exon 51 during Pre-mRNA splicing, restoring the downstream reading frame, and producing a truncated but partially functional dystrophin protein, achieving therapeutic effects.

On December 23, 2016, Spinraza (nusinersen) was approved by the FDA for the treatment of spinal muscular atrophy (SMA). SMA is caused by loss-of-function mutations in the SMN1 gene. Humans have a copy of the SMN1 gene called SMN2, which can encode an unstable protein without exon 7 (SMNΔ7). This drug specifically and stably binds to the intron splicing silencer (ISS-N1) downstream of exon 7 of SMN2 Pre-mRNA, preventing exon 7 of SMN2 from being spliced, resulting in an increase in the amount of SMN2 mRNA transcripts containing exon 7 and the amount of full-length SMN protein, thus achieving the desired therapeutic effect. In February 2019, Spinraza (nusinersen) was approved for marketing in China, becoming the first drug in China to treat SMA.

In 2018, Tegsedi (inotersen) was approved for marketing, used for adult patients with hereditary transthyretin amyloidosis (hATTR) to treat their first or second stage polyneuropathy. Tegsedi can inhibit the production of TTR protein, including both mutant and wild-type forms. The clinical benefits demonstrated by Tegsedi are related to the significant reduction of thyroxine (TTR) protein, which is the underlying cause of hATTR amyloidosis. Tegsedi is associated with the risk of thrombocytopenia and glomerulonephritis, requiring enhanced monitoring to support early detection and management of these risks.

In 2019, Waylivra (volanesorsen) was approved for marketing as a treatment for familial chylomicronemia syndrome (FCS), a rare genetic disease caused by impaired lipoprotein lipase (LPL) function. Patients with FCS may experience severe hypertriglyceridemia (HTG) and an increased risk of triglyceride (TG)-induced pancreatitis. Waylivra can prevent the production of apolipoprotein C-III, slow down fat breakdown, reduce triglyceride levels in the blood, thereby reducing the accumulation of fat in the body, and ultimately lowering the risk of pancreatitis.

On December 12, 2019, Vyondys 53 (Golodirsen) was approved for accelerated marketing by the US FDA for the treatment of patients with Duchenne muscular dystrophy (DMD) who have been confirmed to have exon 53 skipping gene mutations.

On August 12, 2020, the FDA approved the use of Viltepso (viltolarsen) injection for exon 53 skipping therapy in treatable Duchenne muscular dystrophy (DMD) patients. Since the lack of dystrophin is the underlying cause of DMD, increasing dystrophin as early as possible is a key goal in the treatment of DMD. Viltepso is the first and only exon 53 skipping therapy that has demonstrated an increase in dystrophin in children under four years old.

On February 25, 2021, Amondys 45 (casimersen) was approved for marketing for the treatment of Duchenne muscular dystrophy (DMD) patients with exon 45 skipping gene mutations. Amondys 45 became the third antisense oligonucleotide therapy approved in the United States for RNA exon skipping mutations in DMD, following Exondys 51 (eteplirsen) and Vyondys 53 (golodirsen).

2.siRNA Drugs

siRNA is a typical representative of the RNA interference mechanism, capable of achieving efficient silencing of target proteins. siRNA is a double-stranded RNA composed of sense and antisense strands. The site of action for siRNA is in the cytoplasm. The siRNA in the cytoplasm can complex with the RNA-induced silencing complex (RISC). After complexing, the siRNA undergoes duplex dissociation, with the sense strand leaving the RISC complex, while the antisense strand continues to complex with RISC. The antisense strand contains a base sequence that is complementary to the target mRNA, thus enabling it to guide the RISC complex to bind to the target mRNA. The RISC complex contains the nucleic acid degradation enzyme argonaute, and when the siRNA antisense strand binds to the mRNA, the argonaute enzyme in RISC degrades the mRNA, thereby reducing the translation of mRNA into corresponding proteins. siRNA has tremendous potential for the treatment of diseases caused by abnormal gene expression or gene mutations, such as cancer, viral infections, and genetic diseases. Additionally, because the RNA interference process occurs in the cytoplasm without the need to penetrate the nucleus, this makes the development of siRNA nucleic acid drugs even more appealing.

Currently, there are 5 siRNA drugs approved by the FDA and available on the market. On August 11, 2018, the first siRNA drug, Onpattro (patisiran), was approved by the FDA and officially launched for the treatment of hereditary transthyretin-mediated amyloidosis neuropathy. It is the first siRNA drug and the first gene therapy drug utilizing a non-viral delivery system (LNP delivery system). Its intended use is for the treatment of peripheral polyneuropathy caused by hereditary transthyretin amyloidosis (hATTR). Its mechanism involves silencing the expression of hATTR mRNA, reducing the production of hTTR protein, and gradually decreasing the accumulation of amyloid deposits (hTTR) in peripheral nerves, ultimately achieving the purpose of treating the disease. The clinical route of administration is intravenous infusion, administered once every 3 weeks. The molecular weight of siRNA molecules is approximately 14kD, which exhibits the structural characteristics of biological macromolecules.

In 2019, Alnylam's RNAi therapy, Givlaari (givosiran), was approved for market and became the second RNAi drug to be launched after Onpattro (patisiran), for the treatment of adult patients with acute hepatic porphyria (AHP). Givlaari is an RNAi drug administered by subcutaneous injection, which targets and degrades the mRNA encoding the ALAS1 protein. ALAS1 is an important protease involved in the synthesis of ALA and PBG. Monthly treatment with Givlaari? significantly and durably reduces ALAS1 levels in the liver, thereby lowering the levels of neurotoxic ALA and PBG to normal ranges.

On November 23, 2020, the FDA approved Alnylam Pharmaceuticals' RNAi therapy, Oxlumo (Lumasiran), as the first drug to treat primary hyperoxaluria type 1 (PH1), a rare genetic disease. Oxlumo received orphan drug status and breakthrough therapy designation. Oxlumo is the first drug approved to treat PH1 and the only therapy proven to reduce harmful oxalate levels. Phase 3 clinical data showed that Oxlumo treatment significantly reduced oxalate production in the liver, which could potentially address the underlying pathophysiological issues of PH1.

On December 11, 2020, Novartis Pharma GmbH announced that the European Union had officially approved the company's Leqvio (inclisiran) as an adjunct to dietary control for the treatment of adult primary hypercholesterolemia (heterozygous familial and non-familial) or mixed dyslipidemia. Leqvio? became the world's first and only small interfering RNA (siRNA) cholesterol-lowering (LDL-C) therapy, a pioneering "first-in-class" treatment drug globally, marking a significant milestone.

On June 14, 2022, the FDA approved Amvuttra (vutrisiran) for the treatment of polyneuropathy in adults with hereditary transthyretin-mediated (hATTR) amyloidosis. hATTR amyloidosis is a rare, genetic, rapidly progressive, and fatal disease that manifests as polyneuropathy, with few treatment options available.

3.mRNA Drugs

mRNA drugs are composed of hundreds to thousands of nucleotides and can upregulate the expression of target proteins in the body. mRNA functions in the cytoplasm by binding to ribosomes for target protein translation. The target proteins translated by mRNA can be intracellular proteins or secreted by cells to be taken up by other cells (such as antigen-presenting cells) or interact specifically with targets on the cell surface. Although mRNA has poor stability both in vivo and in vitro, mRNA drugs do not require nuclear localization, thus avoiding mutations caused by genomic integration. Therefore, these drugs are considered highly safe.

The application of mRNA nucleic acid vaccines in COVID-19 prevention is a typical representative of mRNA technology. The COVID-19 vaccines Spikevax from Moderna and Comirnaty from Pfizer-BioNTech both belong to mRNA nucleic acid drugs. The bivalent booster versions of these two vaccines have been approved and launched by the US FDA. The mRNA contained in these vaccines encodes the surface spike protein of the novel coronavirus, and the expressed antigen protein is conformationally limited before fusion through S-2P technology to facilitate the presentation of the native antigenic epitopes of the viral surface spike protein. The first approval of these two mRNA COVID-19 vaccines heralded the beginning of the application of mRNA technology drugs in the real world, marking a significant milestone.

The key to the success of these two mRNA COVID-19 vaccines lies in their ability to not only induce the body to produce neutralizing antibodies against the spike protein of the novel coronavirus, but also to generate a robust antigen-specific cellular immune response. The generation of cellular immunity, especially CD8+ T cell immune response, depends on the escape of nucleic acid lysosomes mediated by lipid nanoparticles (LNPs). With the assistance of LNPs, mRNA escapes from lysosomes and enters the cytoplasm of immune cells, where it utilizes ribosomes to express the spike protein. The expressed spike protein is then degraded into fragments by proteasomes in the cytoplasm. In the cytoplasm, these antigen fragments can be recognized by MHC I molecules and presented to T cells, promoting the differentiation of antigen-biased T helper cells and ultimately inducing the maturation of CD8+ T cells.

On the other hand, the spike protein expressed by mRNA can be secreted outside the cell and taken up by other surrounding immune cells, achieving MHC II molecular antigen presentation through the phagosome-lysosome pathway, thereby inducing subsequent humoral immunity dominated by B cells. Both humoral and cellular immunity are equally important in preventing viral infection and disease. In humoral immunity, neutralizing antibodies produced by B cells can specifically bind to free viral spike proteins entering the body, eliminating them through viral particle encapsulation and immune cell phagocytosis. In cellular immunity, CD8+ T cells can recognize cells already infected with the novel coronavirus and eliminate infected cells by releasing strategies such as granzymes and perforins. These two mechanisms, especially the strong cellular immunity induced by mRNA-LNP COVID-19 vaccines through phagosome-lysosome escape, make them show significant advantages over other vaccines in resisting novel coronavirus infection.

4.Nucleic Acid Aptamers

Nucleic acid aptamers are single-stranded nucleic acids with a length of 50 to 120 bases, including DNA aptamers and RNA aptamers. Although they are composed of DNA or RNA like other nucleic acids, the targets of aptamers do not reside on pre-mRNA or mRNA. Instead, they directly act on target proteins and block their functions through spatial hindrance. Compared to antibodies, aptamers have advantages such as simpler synthesis, lower cost, and a wider range of targets, making them have broader potential for drug applications in disease diagnosis, treatment, and prevention.

Currently, the most successful aptamer drug used in disease treatment is pegaptanib sodium, also known as Macugen, which is used to treat age-related macular degeneration (AMD), one of the major causes of blindness in developed countries. AMD is associated with abnormal increases in vascular endothelial growth factor (VEGF), and VEGF165 is the primary isoform of VEGF and the most important therapeutic target for this disease. Pegaptanib sodium can specifically inhibit the activity of VEGF165 by targeting and binding to its heparin-binding domain, preventing VEGF165 from binding to VEGF receptors and thus rendering VEGF165 inactive, achieving therapeutic effects. However, this drug has been withdrawn from the market.

References:

[1] Cui Lili, Zhang Yong. Research Progress on Listed Nucleic Acid Drugs and Their Lipid Nanoparticle Delivery Carriers [J]. Chinese Pharmaceutical Journal, 2023, 58(04)

[2] Wang Jun, Wang Lan, Lv Jiazhen, et al. Analysis of the Therapeutic Effectiveness and Research Progress of Listed Nucleic Acid Drugs [J]. Chinese New Drugs Journal, 2019, 28(18)

[3] Dong Zhihui, Xu Xiaoding. Research Progress and Challenges in Nucleic Acid Drugs [J]. Journal of Practical Medicine, 2022, 38(11)

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.