Introduction:

RNA interference (RNAi) is an endogenous cellular process in which double-stranded small interfering RNA molecules (siRNAs) bind to a specific mRNA target and trigger its degradation, resulting in reduced protein levels in the cell. This gene silencing method allows researchers to study the funciton of proteins within biologcial pathways by transiently removing them and analyzing the impact on cellular function.

Gene transcription control is also mediated using other endogenous RNA molecules, such as microRNAs (miRNAs) and small nuclear RNAs (snRNAs).

While RNA i has proen to be an effective tool for transient gene control, the discovery of CRISPR has revolutionized the field of permant gene editing. The CRISPR-Cas9 system can be used in various gene editing applicaitons and as gene knock-out thorugh the creation of stop condons or splice site variants. CRISPR is often used to validate gene function data that was initially obtained using RNAi approaches. CRISPR relies on the creation of a DNA double strand break and homology directed repair or the less desirable nonhomologus end joining for egene editing to occur.

RNA interference (RNAi):

The term RNA interference (RNAi) was coined after the discovery that injection of dsRNA into the nematode Caenorhabditis elgegans leads to specific silencing of genes highly homologous in sequence to the delivered dsRNA. RNAi was also observed subsequently in insects and other animals. The natural function of RNAi appears to be protection of the genome againsnt invasion by mobile genetic elements such as transposons and viruses, which produce aberant RNA or dsRNA in the host cell when they become active. Specific mRNA degradation prevents transposon and virus replication. DsRNA triggers the specific degradation of homologous RNAs only within the region of identity with dsRNA. (Tuschl “RNA interference is mediated by 21- and 22-nucleotide RNAs” Genes & Development, 2001).

RNA interference (RNAi) is used by eukaryotic cells in a variety of organisms like fungi, plants, worms, mice and probably humans. In plants, RNAi protects cells against RNA viruses. In other types of organisms it may protect against the proliferation of transposable elements that replicate via RNA intermediates. The presence of free, double stranded RNA triggers RNAi by attracting a protein complex containing an RNA nuclease and an RNA helicase. This complex cleaves the double stranded RNA into small fragments. The bound RNA fragments then direct the enzyme complex to other RNA molecules that have complementary nucleotide sequences which can be single or double stranded and the enzyme degrades these as well. In this way, introduction of a double stranded RNA molecule can be used to inactivate specific cellular mRNAs. Thus, RNA interference is typically a two step process. In the first step, input dsRNA is digested into 21-23 nucleotide (nt) small interfering FNAs (siRNAs), probably by the action of Dicer, a member of the RNase III family of double-strand-specific ribonucleases, which cleaves double stranded RNA in an ATP-dependent manner. Successive cleavage events degrade the RNA to 19-21 bp duplexes (siRNA), each with 2-nucleotide 3′ overhangs. In the second step, siRNA duplexes bind to a nuclease complex to form the RNA-induced silencing complex (RISC). An ATP dependent unwinding of the siRNA duplex is required for activation of the RISC. The active RISC (containing a single siRNA and an RNase) then targets the homologous transcript by base pairing interactions and typically cleaves the mRNA into fragments of about 12 nucleotides, starting from the 3′ terminus of the siRNA.

siRNAi:

siRNAs are double stranded RNAs with a MW of about 13kDa which suppress protein translation by recruiting RISC to mRNA via Watson-Crick base pairing. Through the action of the catalytic RISC protein Ago2, a member of the Argonaute family, the target mRNA is cleaved. Alternativley, other Ago proteins (Ago 1, Ago3 and Ago4) catalyse endonuclease mediated nonspecific mRNA degradation by localizing the bound mRNA in processing (P) bodies. (Dahlman “Drug delivery systems for RNA therapeutics” Nature Reviews, Genetics, 23 (May 2022))

siRNA mediated gene silencing has been used safely in humans. These double-stranded RNAs with a MW of about 13kDa suppress protein translation by recruiting RISC to mRNA via Watson-Crick base pairing. Through the action of the catalytic RISC protein Ago2, a member of the Argonaute family, the target mRNA is cleaved. Alternatively, other Ago proteins (Ago1, Ago3 and Ago4) catalyse endonuclease-mediated nonspecific mRNA degradation by localizing the bound mRNA in processing (P)-bodes. siRNA can reduct the expression of any protein-coding gene and has been approved by the FDA and EMA in the form of drugs such as patisiran, which is used to treat hereditary transthyretin-mediated amyloidosis (hATTR), givossiran, which is used to treat acute hepatic pophyria, lumasiran, which is used to treat primary hyperoxaluria type 1 and inclisiran, which is used to treat hypercholesterolaemia. Given that siRNA intefers with mature mRNA, it requires only cytoplasmic delivery, which is easier to acheive than nuclear delivery. (Dahlman, “Drug delivery systems for RNA therapeutics” Nature Reviews Genetics, 23, May 2022).

Antisense Oligonucleotides (ASOs):

Companies: Sarepta Therapeutics 

ASOs are a second class of RNA therapeutics, and are oligonucleotides with a MW of 6-9 kDa. ASOs have the same manufacturing advantages as siRNA and have been approved by the FDA to treat familial hypercholesterolaemia, hATTR amyloidosis with polyneuropathy, specific subtypes of Duchenne muslar dystrophy, and infantile-onset spinal musclular atrophy. ASOs can act throguh three mechanisms of action. First, similar to siRNAs, ASOs bind mRNA via Watson-Crick base pairing, but unlike siRNAs, the ASO DNA-RNA heteroduplex recruits RNase H1 rather than RISC. RNase H1 dependent ASOs are also known as gapmers and lead to cleavage of the target RNA. Second, ASOs can also interfer with splicing machinery by interacting with pre-mRNA, thereby promoting alternative splicing, and increasing target protein expression. Thus, unlike siRNA, which silences target genes, ASOs can be used to increase protein activity in diseases including Duchenne muscular dystrophy and spinal muscular atoprhy. (Dahlman, “Drug delivery systems for RNA therapeutics” Nature Reviews Genetics, 23, May 2022).

MicroRNA (miRNAs): 

MicroRNAs (miRNAs) recruit RISC to complementary mRNA sequences, thereby facilitating targeted RNA interference. As a result, miRNA mimics, which are desigend to increase antive miRNA activity, and ant8-miRNAs or antago-miRNAs, which inhibit miRNA activity, have been studies in animal models and used in clincial trials. Dahlman “Drug delivery systems for RNA therapeutics” Nature Reviews, Genetics, 23 (May 2022)

In 2001, several groups used a cloning method to isolate and identify a large group of miRNA from C. elegans, Drosophila and humans. Several hundeds of miRNAs have been identified in plants and animals which do not appear to have endogenous siRNAs. Thus, while similar to siRNAs, miRNAs are distinct. miRNAs thus far observed have been about 21-22 nucleotides in lenght and they arise from longer precursors, which are transcribed from non-protein-encoding genes. The precursors form structures that fold back on themselves in self-complementary regions; they are then processed by the nuclease Dicer in animals or DCL1 in plants. miRNA molecules interrupt translation through precise or imprecise base-pairing with their targets. Studies have shown that expression levels of numerous miRNAs are associated with various cancers (US 2009/0175827).

Animal cells ahve been shown to express a range of about 22 nucleotide noncoding RNAs termed micro RNAs (miRNAs). The human mir-30 miRNA can be excised from irrelvent, endogenously transcribed mRNAs encompassing the predicted 71 nucleotide mir-30 precursor. One common feature of miRNAs is that they all reside within a putative arm of a predicted aobut 70 nt precursor RNA stem-loop. Dicer, an RNase III-tye enzyme, is beleived to be important for the processing of these miRNA precursors into teh about 22 nt mature miRNAs. (Dicer is also involved in siRNA production form longer dsRNAs above). Expression of the mir-30 miRNA specifically blocks the translation in human cells of an mRNA containing artifical mir-30 target sites. Similalary designed miRNAs can also be excised from transcripts encompassing artificial miRNAs precursores and can inhibit the epxression of mRNAs containg a complementary target site. This approach offers a number of important advantages when contrasted with siRNAs including transfection of miRNA expression plasmids in simple and inexpensive which can result in continous miRNA production. Inhibitor miRNAs could aslo be expressed using viral vectors thaus allowing the production of miRNAs in primary cells or in other cells that are not readily transfectable with syntehtic siRNAs. As the inhibitory RNA is expressed as part of an mRNA, it should also be possible to use regulatable promoters to control miRNA production. (Zen, Molecular Cell, 9, 1327-1333, June 2002).

It is thought that bout 30% of the total genes of the human genome are regulated by miRNAs. The miRNAs are generated through transcription of individual genes in the non-coding regions. The miRNA is transcribed from a pri-miRNA which is a precursor transcribed in the nucleus by RNA polymerase II. The pri-miRNA is cleaved by the RNase III enzyme called Drosha (dsRNA-specific ribonuclease) to produce a pre-miRNA having a hairpin loop structure. The hairpin loop of the pre-miRNA is exported out of the nucleus by the protein exportin-‘5 and Ran-‘GTP, which serve as cofactors, and processed into a miRNA duplex about 22 nucleotides in lenght by the action of teh RNase III enzyme Dicer and TRBP (transactivation-‘responsive RNA binding protein). The miRNA duplex binds with RISC (RNA-‘induced silencing complex) and regulates genes by cleaving mRNAs or preventing translation.

Various kinds of miRNAs and target genes regulated thereby may be useful in predicting the mechanisms of various diseases. Since abnormally increased or decreased miRNA expression is observed in various diseases such as cancer, diabetes and cardiovascular diseases, the miRNA is recognced as a biomarker for diagnosing and predicting diseases.

One phase I clinical trail investigated the use of MRX34, which uses liposomes to deliver a double stranded miRNA-34a mimic, for the treatment of advanced solid tumours. In a phase II clinical trail, the anti-miRNA-122 miravirsen, which binds miRNA-122 and leads to its subsequent inactivity, was tested for the treamtent of hepatitis C. (Dahlman “Drug delivery systems for RNA therapeutics” Nature Reviews, Genetics, 23 (May 2022).

Small Nuclear RNAs (snRNAs):

snRNAs contribute to pre-mRNA splicing regulation rather than binding to mRNA and cuasing degradation. These RNA molecuels are also avialbe as synthetic gene modulators and are able to up or down regulate protein expression transienty.