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RNAi and its Role in Therapeutics
RNA interference (RNAi) as a therapeutic strategy.

Forward genetics has proved to be useful in the detection of the function of the genes in the early days with the help of the knowledge about the phenotype of the mutant gene. However, with the development of technology, reverse genetics has proved to be a better and effective method for the discovery of gene function due to the development of genome sequencing technology, which helped in the discovery of a number of genes without knowing their function. The general method of homologous recombination mediated gene targeting for reverse genetics proved costly, which led to the development of other approaches such as Antisense technology using antisense oligonucleotides as well as the use of ribozyme technology, which also has only limited utilities. Research related to the development of other methods for reverse genetics led to the discovery of the small interfering RNA technology, which showed great promise in revolutionalizing the approach of reverse genetics by knocking down the expression of a particular gene in the vertebrate cells. This technology of using double stranded RNA (dsRNA) to silence specific genes is known as the RNAi technology.

RNA interference involves two types of RNA molecules-micro RNA (miRNA) and small interfering RNA (siRNA). The RNAs are direct products of genes, which produce specific mRNAs by transcription. The siRNAs are duplex RNA molecules 20-25 nucleotides long, which are formed from the long dsRNA by enzymatic cleavage catalysed by Dicer, cytoplasmic RNaseIII enzyme. One of the strands of this siRNA known as the passenger strand is degraded, while the other strand known as the guide strand becomes a part of the RISC (RNA-induced silencing complex) or iRNP complex. It then base pairs with the complementary sequence of the mRNA molecule within the cell and induces its cleavage by the catalytic part of the complex, Argonaute proteins that are actually endonucleases. In this way, the siRNAs prevent the translation of the mRNAs by initiating their degradation. This process is known as RNA interference, which is a posttranscriptional event.

The Pathway of RNAi has some salient features such as the involvement of dsRNA and highly efficient and potent silencing of specific genes with minimum effort that can be introduced in various developmental stages. It has also found application in the identification of different components of various cellular pathways by systemic gene silencing, hence can be used for the development of various targeted and personal therapeutics. RNAi approach has also been useful in cancer therapy as it helps in the knocking down the expression of the anti-apoptotic genes or the cell cycle genes. Research studies have proved the presence of miRNAs in the fragile areas of genome associated with cancer, hence miRNAs may have role in tumor suppression. In some cases miRNAs may function as oncogenes as some studies have found the association of miRNA mutation with various cancers. Studies have shown that some miRNAs may bind to complementary regions in the promoter and may upregulate the expression of some genes, though it has not been clearly illustrated. Hence, miRNAs are also known as Oncomirs, due to their role in cancer.

RNAi has important role in generating immune response against different viruses and has found application in the prevention of self-propagation of the transposons in plants. The novelty of RNAi approach using endogenous mechanism shows great promise in the field of functional genomics that is spreading into the therapeutics. In future, it may offer therapeutics for different metabolic diseases including diabetes, various neurodegenerative diseases, and cardiovascular diseases, which originate from the faulty expression of tissue-specific genes. Hence, it has become a valuable and effective research tool for the biotechnological studies of both living organisms and cell cultures.

However, there are two main challenges in the development of RNAi as therapy. Firstly, the off target effects of RNA interference must be avoided for which in-depth study of the different mechanisms leading to non-specific effects of siRNA is needed and secondly, the efficient delivery of the synthetic siRNAs or iRNA into the specific cell or tissue is very essential for proper development of therapeutic utility of the RNA interference. The use of viral vectors in the delivery of the therapeutic siRNAs into the specific tissues or cells has some safety concerns, which has prompted detailed delivery-related research. RNAi has also found application in the genome wide high-throughput screening of the genes responsible for loss of function as well as genes responsible for specific biological phenotypes. Hence, due to great potential of RNAi in therapeutics, pre-clinical study using this technology is becoming essential.
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Small interfering RNA against ENaC and cystic fibrosis

The use of small interfering RNA technology is being considered in a number of different pathological conditions. Cystic fibrosis is a devastating autosomal recessive disease caused by mutations in the CF transmembrane conductance regulator (CFTR) gene. CF incidence is about 1 in 2000-3000 new births in the European Union; incidence in Ireland is the highest in the world. Incidence in the USA is approximately 1 in 3500 births while in Asia the disease is severely underdiagnosed according to WHO, although existing evidence indicates that it is low. It affects most notably the lungs but also the liver, pancreas and intestines and involves a defect in epithelial transport of sodium and chlorine. Dehydration of the airway surface liquid and build-up of a thick layer of mucus provide the ideal conditions for bacterial biofilm formation. Common bacterial strains found in these biofilms are notably Pseudomonas aeruginosa and also Staphylococcus aureus. The epithelial surface of the airways becomes dehydrated, partially due to hyperactivity of epithelial sodium channels (ENaC), which has been implicated in CF pathogenesis. Patients are locked in a cycle of infection and antibiotic treatment and life expectancy is approximately 40 years. Research into new therapies is urgently needed.

A recent study from the Therapeutic Oligonucleotide Discovery Performance Unit of GlaxoSmithKline (GSK) has identified screened a series of chemically modified 21-mer siRNAs targeting human ENaC . The study identified a siRNA called GSK2225745 which potently inhibited ENaCα mRNA and protein levels in A549 human lung carcinoma cells. GSK2225745 was further tested and shown to exert prolonged inhibition of expression and function of ENaCα in a number of cell lines relevant to CF pathology including bronchial epithelial and nasal epithelial cells. In vivo studies on mice used nanoparticle technology to deliver GSK2225745 to the airways, resulting again in potent inhibition of ENaCα. Further testing of GSK2225745 as a therapeutic agent for cystic fibrosis is indicated.


CLARK, K.L. et al., 2013. Pharmacological Characterization of a Novel ENaCα siRNA (GSK2225745) With Potential for the Treatment of Cystic Fibrosis. United States: Nature Pub. Group
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