Applications of Therapeutic Nucleic Acid and Nucleic Acid Analogs

Therapeutic nucleic acids (TNAs) are nucleic acids themselves or closely related compounds used to treat diseases. Although various types of TNAs exist, they share a common mechanism of action that is mediated by sequence-specific recognition of endogenous nucleic acids through Watson–Crick base pairing. Therapeutic nucleic acids (TNAs) and their precursors are used to treat a variety of diseases and infections. TNA-based therapies have different basic principles and mechanisms and can be divided into three broad categories: 1) therapeutic nucleotides and nucleosides; 2) therapeutic oligonucleotides; 3) therapeutic polynucleotides. Although therapeutic nucleotides and nucleosides that interfere with nucleic acid metabolism and DNA polymerization have been successfully used as anti-cancer and antiviral drugs, they often produce a toxic secondary effect associated with dosage and continuous use. The use of oligonucleotides such as ribozymes and antisense oligodeoxynucleotides (AS-ODN) has shown promise as a therapeutic moiety but faces several problems such as nuclease sensitivity, off-target effects, and efficient delivery. A newer set of therapeutic oligonucleotides, aptamers, is rapidly evolving into early detection and treatment options and has reached commercial interest. In addition, small interfering RNAs (siRNAs) have very high in vitro efficiencies, but they have problems with intracellular target accessibility, specificity, and delivery. Despite the many problems, the precise specificity and versatility of therapeutic oligonucleotides have attracted a great deal of research and resources that will certainly translate them into TNA for the treatment of cancer and viral diseases in the near future.

DNA Therapeutics

• Anti-sense oligonucleotides (ASOs) and DNA aptamers

ASO is a single short-stranded sequence of 8-50 base pairs in length that binds to the target mRNA by standard Watson-Crick base pairing. Upon binding of ASO to mRNA, the target complex will be degraded by endogenous cellular RNase H or functional blockade of mRNA due to steric hindrance. Aptamers (from Latin aptus, suitable), also known as "chemical antibodies", are single-stranded synthetic DNA or RNA molecules of 56-120 nucleotides in length that bind to nucleosides encoding proteins with high affinity acid, therefore used as a bait. DNA aptamers are short single-stranded oligonucleotide sequences similar to ASO and have a very high affinity for target nucleic acids through structural recognition. DNA aptamer, which targets nucleotides for lysozyme, thrombin, human immunodeficiency virus trans-acting response element, hemin, interferon gamma, vascular endothelial growth factor, prostate-specific antigen, dopamine and heat shock factors are still under development. Aptamers are isolated from a large number of nucleic acids by ligand-enriched ligands known as coefficient evolution or SELEX. AptaBid is another selection process that produces appropriate aptamers. They represent an attractive alternative to monoclonal antibodies because they are non-toxic, non-immunogenic, tissue permeable, simple to make, and can be easily modified in vitro, either intravenously or subcutaneously.

Figure 1. ASO mechanism of action⁵

• Gene therapy

Gene therapy replaces the function of an abnormal or non-functional gene with a functioning variant. Gene therapy now covers genetic disorders such as adenosine deaminase deficiency, α-1-antitrypsin deficiency, cystic fibrosis, familial hypercholesterolemia, Gaucher’s disease and hemophilia B. The desired gene is delivered with the aid of a vector, which is usually a virus - most commonly a retrovirus or rarely adenovirus - as a therapeutic gene expression cassette. A therapeutic gene expression cassette is typically composed of a promoter that drives gene transcription, the transgene of interest, and a termination signal to end gene transcription. Table 1 lists some gene therapy clinical trials for primary immunodeficiencies.

Table 1. Gene therapy clinical trials for primary immunodeficiencies⁷

RNA Therapeutics

• RNA aptamers and RNA decoys

A facet of RNAs that makes them promising as therapeutic agents is the ability of some small RNAs to fold into threedimensional structures which can then bind to proteins and thereby inhibit them in a manner similar to protein antagonists. This is the logic behind the use of RNA ‘decoys’ or RNA aptamers. They can bind viral proteins and thus can also be used as vehicles to transport siRNAs. RNA aptamers are developed by methods similar to those described above for DNA aptamers. RNA aptamers are highly susceptible to exonuclease degradation and so require capping with modified or inverted nucleotides such as 2’-O-modified pyrimidines, 2’-amino pyrimidines or 2’-fluoro pyrimidines to prevent terminal degradation. The advantage of RNA aptamers lies in both their ability to reach intracellular targets and also to bind directly to extracellular targets, unlike other RNA-based therapeutics that must first enter the cell to carry out their functions.

The general process of therapeutic RNA-based in mRNA immunotherapy is shown in the figure 2. First, 2-4 hours of hospital leukocyte isolation, monocyte isolation, 6 days of cell culture into in vitro immature DC cells, tumor cells RNA injected into DC cells by electroporation, one day later become mature DC cells, and then Mature DC cells are injected into the patient. The principle is that an RNA molecule vaccine encoding a target antigen is directly injected and absorbed into an antigen presenting cell to induce an immune response in a patient, and the produced effector T cells have a function of targeting attack tumor cells, thereby achieving therapeutic purposes. In animal studies it has been shown that transient antigen presentation results in a long-term anti-tumor immune response. Phase 1 clinical trials have been performed on different types of cancer patients.

Figure 2. Process of therapeutic RNA-based in mRNA immunotherapy¹³

• Ribozymes

An RNA subset that catalyzes RNA or ribozymes can act as an enzyme in the absence of protein. Due to their high specificity, extensive target selection and pre-translational effects, both naturally occurring and synthetic ribozymes can be used to specifically inhibit gene function. They can also be used to validate disease-associated genes as potential targets for new therapeutic interventions. Their ability to cleave mRNA to prevent protein synthesis allows them to find applications in cancer and virology. Among several different types of ribozymes, hammerhead and hairpin ribozymes have been extensively studied. The former is a small ribozyme with specific and catalytic effects and is therefore probably the most widely studied.

At present, for the characteristics of HIV regulatory genes and structural genes, a variety of hairpin and hammerhead anti-HIV-1 ribozymes have been obtained by artificial design and random selection of ribozyme libraries, some of which have entered the first stage of clinical practice. In addition, ribozyme drugs with antiviral effects have been designed for the viral characteristics of HBV and HCV.

Nucleoside Analogs

Nucleoside analogs are a hot spot in the research of antiviral drugs in recent years. The nucleoside analog has an antiviral effect by phosphorylation into a nucleoside analog of triphosphate in the human body and can inhibit the activity of viral DNA polymerase and reverse transcriptase, and compete with the nucleoside for the DNA strand of the virus. The elongation and synthesis of the DNA strand are terminated, and the replication of the virus is inhibited to exert an antiviral action. A representative drug is a lamivudine (dimoxydin, 3TC), which is a newly synthesized dideoxycytidine nucleoside analog, which was first used to treat AIDS. It has been found to have significant anti-HBV effects in patients with mixed HBV and HIV infection.

References

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