RNA interference (RNAi) is a key biological process that leads to the silencing of gene expression. RNAi can be initiated by a cell in response to pathogenic nucleic acids to bring about a targeted degradation of selected genetic transcripts.
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RNAi can also be used experimentally in many scientific applications such as in the elucidation of key cellular processes in health, as well as used therapeutically to inhibit virus function both in vitro and in vivo.
What is RNA Interference?
RNA interference, or RNAi, is a biological process in which RNA inhibits gene expression. Typically, RNAi occurs in response to foreign DNA and double-stranded RNA (dsRNA); which is a hallmark of parasitic and pathogenic (viral) RNA. RNAi is a highly conserved biological mechanism occurring in all eukaryotic cells including plants.
Within a cell, several enzymes and proteins are involved in RNA processing and can direct the RNAi pathway to occur. Firstly, the trigger RNA is processed into a short interfering RNA (siRNA) by Dicer and Drosha (both RNAse II enzymes). Secondly, these siRNAs are loaded into an RNA-induced silencing complex (RISC).
Once the siRNA is assembled into a RISC, the RNA structure is unwound leaving a single-stranded RNA (ssRNA) template that is then able to bind with the mRNA target. Finally, the targeted mRNA undergoes nucleolytic degradation by Argonaute 2 (AGO2), or to gene silencing by direct inhibition of translation.
Experimentally, RNAi is an incredibly valuable research technique that can induce targeted gene suppression of particular genes of interest (reverse genetics); either in cells or animals, to cause a temporary loss-of-function.
Therapeutically, RNAi has wide practical clinical implications for viral infections, cancers, and neurological diseases such as Alzheimer’s disease – although these require more research at this stage before becoming effective treatments.
Furthermore, RNAi can be utilized for large-scale screening repressing individual genes to help elucidate mechanisms underpinning key cellular processes in health and disease. In this article, only RNAi against respiratory viruses will be discussed.
RNAi and Respiratory Viruses
Vaccines against specific viruses – especially respiratory coronaviruses – are often ineffective due to the high number of mutations that occur to such viruses. Antiviral treatments often present with their issues such as antiviral drug resistance.
Respiratory syncytial virus (RSV) is a negative-stranded RNA virus (a Paramyxoviridae) that causes bronchiolitis and pneumonia in children or older immunocompromised adults. RSV results in repeated re-infections throughout life, and to date, there is no vaccine for RSV. Cell studies have successfully used RNAi against RSV (targeted against its viral fusion and phosphoprotein) resulting in efficient inhibition and prevention of infection.
The influenza virus (an Orthomyxoviridae); which also contains segmented negative-stranded RNA, causes seasonal influenza (flu) across all ages. Unlike many other respiratory viruses, the flu jab offers protection against the most recent strains of the virus and can prevent infection in up to 90% of people under 65 years of age, and up to 40% of older adults.
However, the vaccines need to be constantly reformulated yearly to include the most recently prevalent strains of the virus due to the high antigenic drift that occurs each season. Thus, a previous year’s vaccine will offer very limited protection against this year’s influenza virus. On rare occasions, a high antigenic drift results in a novel influenza strain that can lead to an epidemic, or pandemic in uncontrolled.
In cell studies, inhibition by siRNA of Ran-binding protein 5 (essential to viral life cycle), results in delayed accumulation of viral RNAs in infected cells. Here, siRNAs are directed towards a component of the cellular machinery involved in viral processing, can also be effective RNAi targets.
Coronaviruses are large ‘crown’-studded viruses (Nidovirales) and contain positive-stranded RNA that include many different variants, including endemic cold viruses, SARS-CoV (SARS) and the novel SARS-CoV-2 (COVID-19). Compared to other viruses, coronaviruses undergo frequent mutations resulting in no effective vaccine for any coronavirus to date, thus an effective coronavirus drug is desperately needed.
Clinical trials are currently underway for trialing a vaccine against SARS-CoV-2 in the hope to beat the current ongoing global pandemic of COVID-19 – however, only time will tell the results of this trial.
Studies have shown that synthetic siRNAs directed against SARS-CoV-infected cells can inhibit SARS-CoV replication in vitro. Specifically, siRNAs directed against the spike glycoprotein sequences produce a robust inhibition of viral replication.
The spike protein is thought to be the site of the most robust immune response against coronaviruses as well as the site of neutralizing antibodies. If vaccines fail for SARS-CoV-2, RNAi against the spike domain may be an effective therapeutic strategy – however, more research is needed.
Whilst all the studies above have been successful in vitro, the safe and effective delivery of siRNAs in vivo (and clinically) is needed before treatments are made routine. Indeed, trials of introducing siRNAs against influenza A viruses systemically via an intravenous low-pressure injection was able to prevent and treat the infection. Murine studies have also safely and effectively demonstrated the use of intranasal delivery of siRNAs against pathogenic influenza strains.
Other routes include more locally delivered siRNAs due to the less-invasive nature and also reduced the risk of global adverse reactions. Promisingly, siRNA against the spike protein of SARS-CoV (based on the cell studies) was administered in a rhesus macaque monkey (model of SARS) intranasally and led to the abrogation of SARS-CoV infection in the upper airway epithelial cells. This study therefore also holds promise for SARS-CoV-2 due to the similarities between SARS-CoV and SARS-CoV-2.
In summary, RNAi is a highly conserved biological mechanism that responds to pathogenic viral dsRNA to mount an inhibitory molecular response. Many studies have shown that RNAi can be used therapeutically to target many respiratory viruses in vitro and in vivo, including SARS-CoV and potentially SARS-CoV-2, in the treatment and prevention of infection. More research is needed both at the basic and clinical settings before such therapies are made routine.
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