Gene silencing

Gene silencing is the regulation of gene expression in a cell to prevent the expression of a certain gene. Gene silencing can occur during either transcription or translation and is often used in research. In particular, methods used to silence genes are being increasingly used to produce therapeutics to combat cancer and diseases, such as infectious diseases and neurodegenerative disorders.

Ribozymes are catalytic RNA molecules used to inhibit gene expression. These molecules work by cleaving mRNA molecules, essentially silencing the genes that produced them. Sidney Altman and Thomas Cech first discovered catalytic RNA molecules, RNase P and group II intron ribozymes, in 1989 and won the Nobel Prize for their discovery. Several types of ribozyme motifs exist, including hammerhead, hairpin, hepatitis delta virus, group I, group II, and RNase P ribozymes. Hammerhead, hairpin, and hepatitis delta virus (HDV) ribozyme motifs are generally found in viruses or viroid RNAs. These motifs are able to self-cleave a specific phosphodiester bond on an mRNA molecule. Lower eukaryotes and a few bacteria contain group I and group II ribozymes. These motifs can self-splice by cleaving and joining together phosphodiester bonds. The last ribozyme motif, the RNase P ribozyme, is found in Escherichia coli and is known for its ability to cleave the phosphodiester bonds of several tRNA precursors when joined to a protein cofactor.

As of 2014, the miRBase web site, an archive of miRNA sequences and annotations, listed 28,645 entries in 233 biologic species. Of these, 1,881 miRNAs were in annotated human miRNA loci. miRNAs were predicted to each have an average of about four hundred target mRNAs (causing gene silencing of several hundred genes). Freidman et al. estimate that >45,000 miRNA target sites within human mRNA 3'UTRs are conserved above background levels, and >60% of human protein-coding genes have been under selective pressure to maintain pairing to miRNAs.

Gene silencing techniques have been widely used by researchers to study genes associated with disorders. These disorders include cancer, infectious diseases, respiratory diseases, and neurodegenerative disorders. Gene silencing is also currently being used in drug discovery efforts, such as synthetic lethality, high-throughput screening, and miniaturized RNAi screens.

Gene silencing techniques have also been used to target other viruses, such as the human papilloma virus, the West Nile virus, and the Tulane virus. The E6 gene in tumor samples retrieved from patients with the human papilloma virus was targeted and found to cause apoptosis in the infected cells. Plasmid siRNA expression vectors used to target the West Nile virus were also able to prevent the replication of viruses in cell lines. In addition, siRNA has been found to be successful in preventing the replication of the Tulane virus, part of the Caliciviridae family of viruses, by targeting both its structural and non-structural genes. By targeting the NTPase gene, one dose of siRNA 4 hours pre-infection was shown to control Tulane virus replication for 48 hours post-infection, reducing the viral titer by up to 2.6 logarithms. Although the Tulane virus is species-specific and does not affect humans, it has been shown to be closely related to the human norovirus, which is the most common cause of acute gastroenteritis and food-borne disease outbreaks in the United States. Human noroviruses are notorious for being difficult to study in the laboratory, but the Tulane virus offers a model through which to study this family of viruses for the clinical goal of developing therapies that can be used to treat illnesses caused by human norovirus.

Huntington's disease (HD) results from a mutation in the huntingtin gene that causes an excess of CAG repeats. The gene then forms a mutated huntingtin protein with polyglutamine repeats near the amino terminus. This disease is incurable and known to cause motor, cognitive, and behavioral deficits. Researchers have been looking to gene silencing as a potential therapeutic for HD.

There are several challenges associated with gene silencing therapies, including delivery and specificity for targeted cells. For instance, for treatment of neurodegenerative disorders, molecules for a prospective gene silencing therapy must be delivered to the brain. The blood-brain barrier makes it difficult to deliver molecules into the brain through the bloodstream by preventing the passage of the majority of molecules that are injected or absorbed into the blood. Thus, researchers have found that they must directly inject the molecules or implant pumps that push them into the brain.