By Mark Comer
Ribonucleic acid interference (RNAi) is a process employed by cells to post-transcriptionally regulate gene expression. It relies on two types of RNA: microRNA (miRNA) and small-interfering RNA (siRNA). Synthetically produced versions of RNA molecules have shown great therapeutic promise for treating a variety of medical conditions including bacterial and viral infections, and cancer but the side effects and issues with potency have prevented their more widespread use in a therapeutic capacity (Setten, et al 2019). Administered double stranded RNA molecules degrade into single strand molecules which are necessary to induce silencing (Agrawal et al, 2003). Silencing in human cells is dependent on the formation of an RNA-induced silencing complex (RISC), the primary component being the Argonaute 2 protein which is responsible for cleavage of the targeted mRNA alongside the bound miRNA/siRNA. RNAi has also been exploited for research purposes, with synthetic RNA molecules being utilised to ablate expression of certain genes. RNA interference has proven itself to be a potent tool at regulating genetic expression both in vitro and in vivo without having to resort to more direct and permanent methods such as CRISPR-Cas9.
The endogenous process of RNAi begins with transcription of a non-coding sequence in the genome by RNA polymerase II. Both siRNA and miRNA molecules utilise similar downstream intracellular machinery, their differences arise from their origin and how they are initially processed. Three principles are conserved across organisms with regards to RNAi: dsRNA is the start point, mRNA is degraded in a complementary base-pairing fashion, and the degrading machinery is structurally similar across organisms (Agrawal et al, 2003). Micro RNA molecules are originally transcribed as primary miRNA (pri-mRNA) and form stem-loop structures, whilst siRNA molecules are long dsRNA molecules. Drosha recognises pri-mRNA and associates with DGCR8 to remove the hairpin loops from the pri-mRNA, the result is the production of pre-miRNA. Nuclear export occurs via the protein Exportin-5 (Murchison and Hannon, 2004). In the cytoplasm, pre-miRNA molecules are further processed by DICER, an RNase III enzyme, into approximately 22 nt long molecules. Short-interfering RNAs do not require as much processing but are cleaved by DICER as well. The following steps are common to both siRNA and miRNA molecules. One strand of the RNA duplex is incorporated into the multi-protein complex: RISC, the other strand is typically degraded due to its instability (Krol, et al 2004). Argonaute endonuclease proteins within the RISC, cleave the target mRNA strand, miRNA molecules typically have incomplete base pairing whilst siRNA molecules are highly specific.
Conceptually, use of synthetic miRNA or siRNA would allow for modulation of gene expression on the RNA level. Designing an appropriate method of delivering a construct is key, as degradation by intracellular RNA nucleases could potentially completely ablate the construct’s intended effect and any therapy without a proper delivery system would be ineffective. Short-hairpin (shRNA) constructs are used to circumvent this problem, a vector like a bacterial plasmid is used to avoid degradation as these are then transcribed to produce RNA molecules that act as miRNA leading to more stable gene silencing than by transfecting siRNA molecule alone (Taxman, et al 2010). Alternatively, viral vectors can integrate shRNA into a host genome for long term expression and ablation of the targeted gene (Taxman, et al, 2010). Hence, shRNA approaches have long been used as a research tool, especially to aid in mapping novel cellular pathways. For clinical applications, shRNA and miRNA/siRNA approaches have been used successfully in an antiviral capacity by targeting viral genetic material,
showing a capability to suppress replication of HIV in vitro and in vivo¸ albeit to a more limited degree, (Subramanya et al, 2010), as well as with rotavirus and hepatitis B and C (Lundstrom, 2020).
Like with many other novel approaches, RNAi has been tested for its potential to target cancer. Exploiting the RNAi pathway is thought to be another potential route of targeting undruggable proteins by manipulating their expression on the mRNA level. Similar to other precision medicine approaches, the attractiveness of utilising an RNAi-based therapy is the high level of specificity and relatively robust gene silencing effect. Nevertheless, several siRNA-based therapies are in the early phase of clinical trials with targets such as KRAS (Golan et al, 2015) and VEGF (Cervantes et al, 2011). In spite of the theoretical effectiveness of RNAi-based therapies, only two have been approved for use by European and/or American health regulators, both for treatment of rare liver disorders but these represent an important proof-of-concept. Dysregulation of miRNA has also been an area of interest, as they have been implicated in numerous human diseases including cancer and could potentially represent a therapeutic target themselves (Barata, Sood, and Hong, 2016). An miRNA-based approach has the benefit of being able to target multiple genes in comparison to the highly specific siRNA molecules, and combination therapies consisting of a mix of siRNA, miRNA, and conventional drugs are the subject of ongoing research (Barata, Sood, and Hong, 2016).
Despite their impressive level of specificity and potential longevity, off-target effects, poor potency, and degradation by nucleases represent the key hurdles for effective RNAi-based therapies. Potential solutions mainly revolve around novel delivery systems, predominantly based on nanoparticle delivery systems, to allow for more precise targeting and improved uptake. Whilst RNA interference-based drugs and therapies are currently few and far between it seems that with continued research and optimisation an RNAi platform could become the latest addition to a clinician’s ever-expanding therapeutic toolbox.
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