‘RNA interference’ refers to a defense mechanism developed to counter the invading viruses at the RNA level (Agrawal et al., 2003). The post-transcriptional gene silencing mechanism was originally identified in plants and subsequently shown to be operational in various eukaryotes (ex. fungus, vertebrates). It involves processing a long double stranded RNA (dsRNA) into short 21-25 bp RNA duplexes (with 2 bp overhang at 3’ terminus and phosphorylated 5’ terminus) called ‘small interfering RNAs’ (siRNA) by the enzyme Dicer. The siRNA duplex is presented by Dicer and TAR RNA binding protein (TRBP) to Argonaute to form the ‘RNA-induced silencing complex’ (RISC) (Wilson et al., 2013). After unwinding the siRNA duplex, one strand is selected (the other dissociates from RISC) to serve as a guide strand to recognize and cleave target complementary single stranded RNA. In addition to degrading viral genomic RNA or suppressing the expression of mRNA, RNA interference may function to increase gene expression as targeting a promoter can activate transcription (Li et al., 2008).
The potential of developing the RNA interference-based therapy for various infectious diseases or genetic disorders (ex. neurodegenerative disorders, cancer) is increasingly being explored. The siRNA technology involves introducing short dsRNA (~21 bp in length with 2 nucleotide 3’-overhang) into cells. Upon incorporation into the silencing complex RISC, the sense strand is removed typically. The remaining antisense strand guides RISC to target mRNA. Following the cleavage of the target mRNA at a discrete position, the resulting 5’ fragment and 3’ fragment are degraded by exosome and 5'-3' exoribonuclease 1 (XRN1), respectively. Its catalytic nature allows the degradation process to be repeated with additional mRNA targets. For pharmacological application, various issues such as stability, delivery, renal clearance and immune response are being addressed.
In 2018, Food and Drug Administration (FDA) approved the first RNA interference inducing drug Onpattro. Onpattro (also known as Patisiran) was developed by Alnylam Pharmaceuticals, Inc. to treat hereditary transthyretin-mediated amyloidosis (hATTR), a neurodegenerative disorder causing polyneuropathy (dysfunction of peripheral nerves). This rare but life-threatening disease is caused by the deposition of circulating transthyretin (TTR) amyloid in peripheral nerve, heart, kidney, gastrointestinal tract, etc., causing sensorimotor deterioration and various other symptoms including heart failure. Most hereditary cases are heterozygous for TTR mutation, and both mutant and normal TTR are found in the amyloid deposits (Coelho et al., 2013).
Onpattro (Patisiran) is a 21-mer double-stranded small interfering RNA (siRNA) oligonucleotide that targets 3’-UTR (untranslated region) shared by both normal and mutant TTR mRNAs. It incorporates 2’-O-methylcytidine and 2’-O-methyluridine at specific locations, with 2´-deoxythymidine dinucleotide overhangs at both 3´ ends. The RNA duplex is encapsulated in a cationic lipid nanoparticle, which is opsonized with ApoE (apolipoprotein E) to facilitate binding to ApoE receptor present on hepatic cell surface for uptake via endocytosis (Coelho et al., 2013). Phase 3 clinical trial demonstrated that Onpattro improves the symptoms of adult patients suffering from the hereditary transthyretin amyloidosis (Adams et al., 2018). One caveat is that the prescription cost for Onpattro could be quite high.
These advances have set the stage for the development of RNA interference therapy for other disorders such as cancer, diabetes, cardiac disease, etc. Additionally, the potential of utilizing siRNA oligonucleotides to treat infectious diseases (ex. hepatitis B virus) has been investigated. Previously, for SARS coronavirus, siRNAs targeting the regions in the genome encoding spike protein and ORF1b (NSP12) have been designed (Li B et al., 2005). Another report showed that siRNA targeting the ‘Leader’ sequence could suppress the replication of SARS coronavirus (Li T et al., 2005).
With the event of COVID-19 coronavirus pandemic, there is an avid interest in developing siRNA as prophylactic or therapeutic agent. Alnylam Pharmaceuticals plans to develop siRNA targeting the host factor ACE2 (angiotensin converting enzyme-2; receptor for COVID-19) or TMPRSS2 (transmembrane protease, serine 2; cleaves spike protein to mediate virus entry) to impede the coronavirus infection. One challenge in developing siRNA therapeutic is the existence of virus encoded proteins that suppress RNA interference as have been documented for influenza A virus, vaccinia virus, Ebola virus, etc. In the case of SARS coronavirus, its nucleocapsid protein N may repress RNA interference of the host cell (Cul et al., 2015).
Bio-Synthesis, Inc.—with its capacity to provide siRNA oligonucleotides with unsurpassed specificity and stability--is committed to supporting COVID-19 therapy initiatives. Further, the key to preventing epidemic is the ability to diagnose the infected early to preempt further propagation. For this, it provides primers and probes (as well as synthetic RNA control) for COVID-19 diagnosis via RT-PCR assay. Bio-Synthesis, Inc. also specializes in oligonucleotide modification and provides an extensive array of chemically modified nucleoside analogues (over ~200) including bridged nucleic acid (BNA). A number of options are available to label oligonucleotides (DNA or RNA) with fluorophores either terminally or internally as well as conjugate to peptides. It recently acquired a license from BNA Inc. of Osaka, Japan, for the manufacturing and distribution of BNANC, a third generation of BNA oligonucleotides. To meet the demands of therapeutic application, its oligonucleotide products are approaching GMP grade. Bio-Synthesis, Inc. has recently entered into collaborative agreement with Bind Therapeutics, Inc. to synthesize miR-21 blocker using BNA for triple negative breast cancer. The BNA technology that we offer provides superior, unequalled advantages in base stacking, binding affinity, aqueous solubility and nuclease resistance. It also improves the formation of duplexes and triplexes by reducing the repulsion between the negatively charged phosphates of the oligonucleotide backbone. Its single-mismatch discriminating power was especially useful for diagnosis (ex. FISH using DNA probe). More importantly, BNA oligonucleotide exhibits lesser toxicity than other modified nucleotides for clinical application.
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https://www.biosyn.com/tew/Speed-up-Identification-of-COVID19.aspx
https://www.biosyn.com/covid-19.aspx
https://www.biosyn.com/sirna-overview.aspx
References
Adams D, Gonzalez-Duarte A, O'Riordan WD, Yang CC, Ueda M, et al. Patisiran, an RNAi Therapeutic, for Hereditary Transthyretin Amyloidosis. N Engl J Med. 379:11-21 (2018). PMID: 29972753 doi: 10.1056/NEJMoa1716153.
Agrawal N, Dasaradhi PV, Mohmmed A, Malhotra P, Bhatnagar RK, Mukherjee SK. RNA interference: biology, mechanism, and applications. Microbiol Mol Biol Rev. 67:657-85 (2003). PMID:14665679 DOI: 10.1128/mmbr.67.4.657-685.2003
Coelho T, Adams D, Silva A, Lozeron P, Hawkins PN, Mant T, et al. Safety and efficacy of RNAi therapy for transthyretin amyloidosis. N Engl J Med. 369:819-29 (2013). PMID: 23984729 doi: 10.1056/NEJMoa1208760.
Cui L, Wang H, Ji Y, Yang J, Xu S, Huang X, Wang Z, et al. The Nucleocapsid Protein of Coronaviruses Acts as a Viral Suppressor of RNA Silencing in Mammalian Cells. J Virol 89:9029-43 (2015). PMID: 26085159 PMCID: PMC4524063 DOI: 10.1128/JVI.01331-15
Li BJ, Tang Q, Cheng D, Qin C, Xie FY, Wei Q, et al. Using siRNA in prophylactic and therapeutic regimens against SARS coronavirus in Rhesus macaque. Nat Med 11:944-51 (2005). PMID: 16116432 PMCID: PMC7095788 DOI: 10.1038/nm1280
Li LC. "Small RNA-Mediated Gene Activation". RNA and the Regulation of Gene Expression: A Hidden Layer of Complexity. Caister Academic Press. ISBN 978-1-904455-25-7 (2008).
Li T, Zhang Y, Fu L, Yu C, Li X, Li Y, et al. siRNA targeting the leader sequence of SARS-CoV inhibits virus replication. Gene Ther. 12:751-61 (2005). PMID: 15772689 DOI: 10.1038/sj.gt.3302479
Wilson RC, Doudna JA. Molecular mechanisms of RNA interference. Annu Rev Biophys. 42:217-39 (2013). PMID: 23654304 PMCID: PMC5895182 doi: 10.1146/annurev-biophys-083012-130404.