Gene silencing technologies

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Chapter: Pharmaceutical Drugs and Dosage: Biotechnology-based drugs

Gene silencing technologies can be exemplified by antisense ODNs, peptide nucleic acids (PNAs), antisense RNA, aptamers, ribozymes, and double-stranded siRNA.

Gene silencing technologies

Gene silencing technologies can be exemplified by antisense ODNs, peptide nucleic acids (PNAs), antisense RNA, aptamers, ribozymes, and double-stranded siRNA.

Antisense oligonucleotides

To create antisense drugs, nucleotides are linked together in short chains, called ODNs. When deoxyribonucleotides are linked in small chains, these are called oligodeoxyribonucleotides. The sequence of nucleotides in the antisense drugs is complementary to small segments of mRNA. Each antisense drug is designed to bind to a specific sequence of nucleotides in its mRNA target to inhibit production of the protein encoded by the tar-get mRNA. By acting at this earlier stage in the disease-causing process to prevent the production of a disease-causing protein, antisense drugs have the potential to provide greater therapeutic benefit than traditional drugs—which do not act until the disease-causing protein has already been produced. Antisense drugs also have the potential to be much more selec-tive or specific than traditional drugs, and therefore more effective, because they bind to specific mRNA targets through multiple points of interaction at a single binding site. An oligomer of about 15–20 nucleotides in length is considered to be the best because this corresponds to both the appropriate length of a single unique target site in mRNA and the length required for effective hybridization. Cellular uptake of ODNs occurs by means of fluid-phase pinocytosis and/or receptor-mediated endocytosis.

Figure 26.2 shows the structures of antisense compounds. Unmodified ODNs are polyanions with a phosphodiester backbone. They are rapidly degraded under physiological conditions by enzymes called nucleases, pri-marily 3'-exonucleases. Because of this, ODN modifications have been designed to prevent or reduce the rate of degradation. The phosphorothio-ate modification of the ODN backbone, in which a sulfur atom replaces one of the nonbridging oxygen atoms in the phosphate group, produces ODNs that are relatively resistant to cellular and serum nucleases. Methylphosphonate ODNs have no net charge, which prevents nuclease digestion, but also decreases water solubility (Figure 26.2a).

Figure 26.2 Structures of antisense compounds. (a) antisense oligonucleotide (ODN), (b) peptide nucleic acid (PNA), and (c) antisense RNA. R: –O, phospho-diester oligonucleotide; –S, phosphorothioate oligonucleotide; –CH3, methylphosphonate oligonucleotide.

Triplex-forming oligonucleotides

In contrast to antisense ODNs, triplex-forming oligonucleotides (TFOs) inhibit gene transcription by forming DNA triple helices in a sequence-specific manner on polypurine–polypyrimidine tracts. Targeting TFOs to the gene itself presents several advantages compared to antisense ODNs, which are directed to mRNA. 

There are only two copies of targeted gene, whereas there are thousands of copies of mRNA. Blocking mRNA trans-lation does not prevent the corresponding gene from being transcribed, which continuously repopulates the RNA pool. In contrast, prevention of gene transcription can bring down the mRNA concentration in a more efficient and long-lasting way.

DNA normally exists in a duplex form (two strands coiled around each other). However, under some circumstances, DNA can assume triple helix structures. Triplex helix formation may then prevent the interaction of vari-ous transcription factors, or it may physically block the initiation or elonga-tion of the transcription complex. This process is used in the application of TFOs. The TFOs are specific DNA duplex binding ODNs that can bind to DNA duplex, leading to triple helix formation and prevention of the transcription process.

Peptide nucleic acids

PNA has a chemical structure similar to DNA and RNA but differs in the composition of its backbone. DNA and RNA have a deoxyribose and ribose sugar backbone, respectively, whereas PNA’s backbone is composed of repeating N-(2-aminoethyl)-glycine units linked by peptide bonds. The various purine and pyrimidine bases are linked to the backbone by methylene carbonyl bonds. PNAs are depicted similar as peptides, with the N-terminus at the first (left) position and the C-terminus at the right (Figure 26.2b). As the backbone of PNA contains no charged phosphate groups, the binding between PNA/DNA strands is stronger than between DNA/DNA strands due to the lack of electrostatic repulsion. PNAs are resistant to both nucleases and proteases (enzymes that digest proteins). PNAs can bind to DNA and RNA targets in a sequence-specific manner to form PNA/DNA and PNA/RNA Watson–Crick double helical structures. Therefore, PNAs can be used as antisense medicines similar to ODNs.

Antisense RNA

The antisense mRNA strategy relies on the transfection and subsequent expression of a plasmid vector whose gene expression cassette carries the cDNA of the gene of interest subcloned into the vector in an antisense orien-tation (Figure 26.2c). After transfection (the process of introducing foreign genetic material into cells) into the cells, the plasmid expresses the anti-sense mRNA within the cell cytoplasm. This antisense mRNA can hybrid-ize exclusively with the mRNA of the gene of interest and can block protein synthesis (translation). Hence, antisense mRNA gene medicines require expression vectors and delivery systems similar to gene therapy medicines (discussed later in this chapter).


MicroRNAs (miRNAs) are endogenous, noncoding, small (~22 nucleo-tides) double-stranded RNAs that participate in gene silencing and post-transcriptional regulation of gene expression. The miRNAs regulate several cellular processes to impact overall outcomes in areas such as cell survival and fat metabolism. Each miRNA has multiple targets and makes global changes in the cellular systems. Changes in the expression level of miRNAs are associated with phenotypic or performance differentiation among different cells. For example, high and low titer producing chinese hamster ovary (CHO) cell lines have shown differences in the concentra-tions of specific miRNAs. Manipulating the levels of miRNAs in CHO cells leads to increased recombinant protein production by influencing cell proliferation, increasing cellular resistance to apoptosis, and increasing specific productivity.

Gene medicines that utilize the miRNA pathway might either be exog-enously administered miRNAs or gene silencing therapies that block the production of endogenous miRNAs.


Aptamers are single-stranded or double-stranded nucleic acids that can bind proteins involved in the regulation and expression of genes (i.e., tran-scription factors). In addition, they also bind to proteins that perform other regulatory functions. For example, a 15-mer (i.e., 15 nucleotide long) DNA aptamer binds to human thrombin and prevents thrombin-catalyzed blood coagulation. In this approach, the target site is extracellular, and hence the aptamer nucleic acid does not have to cross the cell membrane to be effec-tive, after parenteral administration.


Ribozyme, also known as RNA enzyme or catalytic RNA, is an RNA mol-ecule having catalytic enzyme activity that uses either transesterification or a hydrolysis mechanism to cleave a unique phosphodiester bond in a single-stranded RNA molecule in a sequence-dependent manner. This process, therefore, leads to interference with the translation process.

RNA interference

RNAi is a phenomenon in which double-stranded RNA (dsRNA) mole-cules efficiently and specifically inhibit gene expression at a posttranscrip-tional level. Endogenous mRNA exists as a single strand. Mammalian cells have a specific enzyme, called Dicer, which recognizes the double-stranded RNA and chops it up into small fragments of between 21–25 base pairs in length. Such a short double-stranded RNA fragment is called siRNA. The siRNA can bind certain cellular proteins to form the RNA-induced silenc-ing complex (RISC). The RISC gets activated when the siRNA unwinds. The activated complex binds to the mRNA corresponding to the antisense RNA. Thus, siRNA silences a target gene by binding to its complementary mRNA and by triggering its degradation. The mechanisms of RNAi are illustrated in Figure 26.3.

Figure 26.3 Mechanisms of RNA interference. Long double-stranded RNA is cleaved by Dicer into fragments of 21–23 nucleotide siRNAs. Following unwinding, the antisense strand of duplex siRNA is incorporated into RNA-induced silenc-ing complex (RISC) protein. Subsequently, the incorporated siRNA stand guides RISC to its homologous target mRNA for endonucleolytic cleavage.

Potent knockdown of the target gene with high sequence specificity makes siRNA a promising therapeutic strategy. Three different ways are commonly used for producing siRNA: chemical synthesis, administration of plasmid DNA, and viral vectors encoding small hairpin RNA (shRNA) expression cassette. The transcription of genetic sequence in the plasmid DNA or viral vector leads to the production of an mRNA that has internal self-complementarity, which leads to the formation of double strand with a closed hairpin-like loop at one end (shRNA). The shRNA becomes a sub-strate for the Dicer, leading to endogenous formation of siRNA.

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