Post-Transcriptional Gene Silencing (PTGS)
Gene silencing, often used in context of RNA interference (RNAi), is a regulatory pathway by which the expression of a gene is suppressed. Although a gene may be silenced at the transcriptional or post-transcriptional stages, RNAi is specifically a post-transcriptional gene silencing (PTGS) mechanism achieved by cellular introduction of a double stranded RNA (dsRNA) termed siRNA or microRNA.
Gene silencing occurs naturally in cells, and is thought to play a large part in the overall regulation of gene expression in many organisms, giving them protection from viral infections and other undesirable elements. Recent advances in gene silencing for basic research have lead to the concept of RNAi therapeutics against incurable diseases including hepatitis, cancer, viral infections and HIV by suppressing the expression of genes responsible for promoting these particular diseases. Gene silencing takes place by degrading the mRNA required for protein synthesis. In the absence of template mRNA, the cell is rendered incapable of producing proteins, thus suppressing expression and availability of the target protein.
The RNAi pathway is present in eukaryotes and is initiated by Dicer, an enzyme which slices long, double-stranded RNA molecules into shorter fragments. Each fragment contains ~20 nucleotides of double stranded RNA (dsRNA) and the guide strand is loaded into an RNA induced silencing complex (RISC). At this point, RISC uses the guide strand as a template to find the complimentary sequence within an mRNA strand. Upon binding, the strand of mRNA is cleaved, which reduces mRNA and subsequent protein expression levels of the target gene. Along with several proteins, this double-stranded RNA forms the RNA-induced silencing complex (RISC) which utilizes the dsRNA as a template to degrade any RNA that shares the sequence of the dsRNA. RISC has been shown to be effective at silencing specific genes.
The selective effect RNAi has on gene expression makes it an invaluable research tool to study mRNA target effects, pathway analysis and PK/PD effects. RNAi enables these studies for both cell cultures (utilizing in vitro siRNA and DNA transfection reagents) and living organisms (in vivo delivery tools, nanoparticle-, polymer-, lipid-and PEG-liposome based in vivo transfection reagents). RNAi has been demonstrated effectively in cells and tissues, including both exogenous and endogenous genes. The resultant effect can be partial or diminishing knockdown of the target gene but not complete silencing of the expression (i.e. 50 to 70% reduction), or near complete silencing or turning off of the target gene (i.e. 95 to 99% reduction). Long dsRNA (e.g. >30 base pairs) can trigger an interferon response which leads to mRNA cleavage and apoptosis, thus the length of the oligonucleotide is a crucial parameter to control in the design and manufacturing of RNAi products.
The RNAi pathway may have originated evolutionarily as an early virus-identification system in cells, enabling the identification and destruction of any double-stranded RNA as an effort to prevent viral replication. Alternatively, one hypothesis is that the RNAi pathway merely controls transcription; small RNAs may determine transcription rates of genes.
Discovery of the RNAi effect won Craig Mello and Andrew Fire the Nobel Prize in Medicine in 2006. Initially, a single strand of RNA was injected into nematodes that bound to its target mRNA. They later discovered that a double-stranded version of the same sequence intensified the observed gene-silencing effect. Their work and others brought forth the full extent to which RNAi can be utilized in an animal system with a strong RNAi therapeutics potential. Today, there are commercial siRNA therapeutic development programs offered by biology CRO companies that provide pre-clinical research services, complete RNAi CRO services, cell banking and generation of stable cell lines.
Prior to extensive knowledge of the RNAi pathway, gene silencing could be accomplished through transference from stock to scion plants by means of grafting. It is believed that RNAi evolved to protect cells from foreign materials, such as viruses, to prevent self-propagation by transposons. An example is in both juvenile and adult Drosophila, where RNAi is shown to be active and play a vital role in anti-virus attack against pathogens such as Drosophila X virus.
The RNAi pathway is triggered when short, double-stranded RNA (dsRNA) is formed. Double stranded RNA can occur several ways:
- Viruses: Introduce dsRNA into the cell
- siRNA: Introduction of small interfering RNA (siRNA), a double-stranded 20-24 bp segment of RNA ending with two overhanging nucleotides and phosphorylated 5′ ends and hydroxylated 3′ ends is administered artificially. The effects of siRNA are transient and last 3-7 days when transfected into an organism.
- Plasmid DNA: Introduction of a plasmid DNA that produces short hairpin RNA (shRNA); a small piece of RNA (typically 19 to 29 base pairs, joined by a 4 nucleotide loop) that is self-complementary and doubles over to appear double-stranded. Dicer recognizes shRNA and cleaves it for conversion into siRNA. shRNA effects can last up to 3 years.
- miRNA: synthetic miRNA mimics are 20-22 bp nucleotide strands of RNA that regulate gene expression by binding to a complementary mRNA strand, thus making the mRNA double-stranded and inhibiting its transcription and translation. miRNA are endogenously expressed in cells but are short-lived when introduced into the cell by transfection.
The RNAi response has demonstrated effective gene regulation in mammalian cells and tissues for both endogenous and exogenous genes. The amount of gene knockdown can be partial or complete, but depends on complicated factors associated with the nature and length of the siRNA.