Small interfering RNA
It interferes with the expression of specific genes with complementary nucleotide sequences by degrading messenger RNA (mRNA) after transcription, preventing translation.
In 1998, Andrew Fire at Carnegie Institution for Science in Washington DC and Craig Mello at University of Massachusetts in Worcester discovered the RNAi mechanism while working on the gene expression in the nematode, Caenorhabditis elegans.
[4] They won the Nobel prize for their research with RNAi in 2006. siRNAs and their role in post-transcriptional gene silencing (PTGS) was discovered in plants by David Baulcombe's group at the Sainsbury Laboratory in Norwich, England and reported in Science in 1999.
[6] In 2001, the expression of a specific gene was successfully silenced by introducing chemically synthesized siRNA into mammalian cells (Tuschl et al.) These discoveries led to a surge in interest in harnessing RNAi for biomedical research and drug development.
[14] One report in the Aedes aegypti mosquito has shown there is some evidence for RNAa and can be achieved by short or long dsRNAs targeting promoter regions.
The mRNA molecule is then cut precisely by cleaving the phosphodiester bond between the target nucleotides which are paired to siRNA residues 10 and 11, counting from the 5'end.
Once considered as "dark matter" of ncRNAs, piRNAs emerged as important players in multiple cellular functions in different organisms.
[21] Many model organism, such as plants (Arabidopsis thaliana), yeast (Saccharomyces cerevisiae ), flies (Drosophila melanogaster) and worms (C. elegans), have been used to study small non coding RNA-directed Transcriptional gene silencing.
This approach has been considered as therapeutically crucial for the silencing dominant gain-of-function (GOF) disorders,where mutant allele causing disease is differed from wt-allele by a single nucleotide (nt).
Furthermore, because structurally related microRNAs modulate gene expression largely via incomplete complementarity base pair interactions with a target mRNA, the introduction of an siRNA may cause unintended off-targeting.
[26] Most evidence to date suggests that this is probably due to activation of the dsRNA sensor PKR, although retinoic acid-inducible gene I (RIG-I) may also be involved.
Chemical modification of siRNA is employed to reduce in the activation of the innate immune response for gene function and therapeutic applications.
[28] MicroRNAs occur naturally, and by harnessing this endogenous pathway it should be possible to achieve similar gene knockdown at comparatively low concentrations of resulting siRNAs.
Some methods for siRNA delivery adjoin polyethylene glycol (PEG) to the oligonucleotide reducing excretion and improving circulating half-life.
[32] Thus, while siRNAs can produce unwanted off-target effects, i.e. unintended downregulation of mRNAs via a partial sequence match between the siRNA and target, the saturation of RNAi machinery is another distinct nonspecific effect, which involves the de-repression of miRNA-regulated genes and results in similar problems in data interpretation and potential toxicity.
[38] siRNA oligos in vivo are vulnerable to degradation by plasma and tissue endonucleases and exonucleases[40] and have shown only mild effectiveness in localized delivery sites, such as the human eye.
[41] Delivering pure DNA to target organisms is challenging because its large size and structure prevents it from diffusing readily across membranes.
[40][43] siRNAs delivered via lipid based nanoparticles have been shown to have therapeutic potential for central nervous system (CNS) disorders.
[45] Phase I results of the first two therapeutic RNAi trials (indicated for age-related macular degeneration, aka AMD) reported at the end of 2005 that siRNAs are well tolerated and have suitable pharmacokinetic properties.
[46] In a phase 1 clinical trial, 41 patients with advanced cancer metastasised to liver were administered RNAi delivered through lipid nanoparticles.
For RNAi to realize its therapeutic potential, small interfering RNA (siRNA) must be delivered to the site of action in the cells of target tissues.
When quick but powerful electrical pulses are initiated the lipid molecules reorient themselves, while undergoing thermal phase transitions because of heating.
This results in the making of hydrophilic pores and localized perturbations in the lipid bilayer cell membrane also causing a temporary loss of semipermeability.
Delivering this siRNA from DNA templates can be done through several recombinant viral vectors based on retrovirus, adeno-associated virus, adenovirus, and lentivirus.
Another advantage is that, unlike small molecule and monoclonal antibodies that need to recognize specific conformation of a protein, siRNA functions by Watson-Crick basepairing with mRNA.
[4] Animal models did not accurately represent the degree of immune response that was seen in humans and despite the promise in the treatment investors divested away from RNAi.
Repeated developmental work also shed light on improving the chemical composition of the RNA molecule to reduce the immune response, subsequently causing little to no side effects.
Target messenger RNA (mRNA) is cleaved as a result by tiny interfering RNAs coupled to the RNA-induced silencing complex.
Givlaari is an siRNA drug that downregulates the expression of aminolevulinic acid synthase 1 (ALAS1), a liver enzyme involved in an early step in heme production.
Several other big pharmaceutical companies such as Amgen and AstraZeneca have also invested heavily in siRNA therapies as they see the potential success of this area of biological drugs.
