Considering the two most common types of diseases caused by mRNA activity, one of them is virus-related and the other is cancer-related1. These diseases cause viral or abnormal proteins to be encoded by mRNA1. For instance, some ORFs found in coronaviruses encode viral structural proteins2. After viral cells enter the body, they undergo RNA translation and infect the organism by producing viral proteins for the next transcription3. Moreover, some of the cancer-associated changes in gene expression are due to functional defects in microRNAs involved in the post-transcriptional regulation of mRNAs4. As a result, mutagenic genes encoding abnormal proteins lead to the formation of cancer diseases such as leukemia1. Therefore, one of the most promising methods developed to prevent these processes is RNA interference (RNAi)1.
RNA interference is the silencing of gene expression by degrading a specific mRNA5. The cells in which RNA interference was first investigated belonged to Caenorhabditis elegans (C. elegans)6. Eventually, using the RNAi method, mRNAs copied from a viral or mutagenic cancer gene can be inhibited, thereby stopping the transcription of this mRNAs1. The first step in RNA interference is the splitting of the double-stranded RNA (dsRNA) into small interfering RNAs (siRNAs) that are 21-23 nucleotides long7,8. The enzyme used in the cleavage of this dsRNA is Dicer9. Secondly, siRNAs are incorporated into a ribonucleoprotein particle called RNA-induced silencing complex (RISC)1,8. The siRNAs are complementary to one of the sequences of the targeted mRNAs1. RISC targets homologous transcript with base pairing after unwinds the siRNA10.

RNA interference therapeutics have been studied preclinically in cells that belong to patients or animal models. With these preclinical studies, it has been revealed that the RNAi method sheds light on the solution to many problems, including viral infections1. For instance, RNAi can block the coding of abnormal proteins that lead to cancer. For example, the BCR-ABL gene, which is formed because of the combination of two normal genes, causes a type of leukemia. In this case, siRNA can turn malignant cells back to normal1. Likewise, the RNA interference method can also be used to inhibit the expression of abnormal huntingtin (HTT) proteins that cause Huntington’s disease. Thus, by decreasing the expression, protein clearance mechanisms within neurons are also improved11.
Furthermore, in some studies, it has been shown that the RNA interference method can prevent diseases caused by deadly viruses such as Human Immunodeficiency virus (HIV), Hepatitis C and B viruses (HCV and HBV), and Influenza A virus (IAV)12. In the case of HIV, stem cells from patients are transfected with vectors carrying the siRNA and then injected back into the patient’s blood. Small interfering RNAs are produced and through this process, cells can resist the attack of viruses1. Also, there is no vaccine for HCV, but the RNAi method has been used to target HCV replication13. Since the use of a cell culture system for HCV replication is very difficult, replicon systems from Huh-7 cells were used in these studies. The researchers used siRNAs and shRNAs (Short hairpin RNAs) to inhibit the replication of HCV13.
In addition to all these promising aspects, there are some points where RNA interference is ineffective in the treatment of viral diseases. One of the biggest problems is that the viruses that cause the disease mutate very quickly and change their gene sequence. Due to changes in genome sequences, therapeutic siRNA cannot complete new mRNA sequences1. The first test of RNA interference technology in humans was carried out on a degeneration that can lead to vision loss in the elderly because of an overgrowth of blood vessels behind the retina. To solve this problem caused by the VEGF growth factor, patients’ eyes were injected with siRNAs targeting this VEGF-encoding mRNA. Although it was initially suggested that these siRNAs have beneficial effects, no results were obtained to support this in the later stages of the trial1. Another RNAi therapeutic test was performed against a respiratory virus (RSV). The subjects inhaled aerosols containing siRNA, and as a result, successful results were obtained1. Another limitation of RNA interference is the need for site-specific delivery of siRNA12. The siRNAs used in RNA interference must reach where they need to be delivered and perform the inhibition there14. To solve this problem, direct chemical modification of siRNA, liposome formulations, and nanoparticles have been developed15. In a study, a protamine-antibody fusion protein was created to deliver siRNA to HIV-infected cells. As a result, an antibody-mediated delivery system specific to siRNAs was created using cell surface receptors16.
To sum up, RNA interference is one of the most promising methods for diseases recently. The basis of this method is the inhibition of transcription of gene sequences encoding abnormal proteins. Small interference RNAs complementary to the problematic gene region are used to achieve this1. While the RNAi method gave positive results in some studies, it did not show any effect in others. Many new ideas continue to be produced, especially for the treatment of viral or cancer-related diseases1. One of these promising approaches is the creation of molecular peptide-siRNA nanostructures that can deliver specific cell types more selectively than the endothelial permeabilization mechanism17. RNA interference technology is still in the early stages of a research journey and although it has some challenges, it is on the way to becoming a great therapeutic method.
Refeences:
- Cell and Molecular Biology by Gerald Karp, 7th Edition
- Liu D.X., Fung T.S., Chong K.K., Shukla A., Hilgenfeld R. Accessory proteins of SARS-CoV and other coronaviruses. Antiviral Res. 2014;109:97–109.
- Nakagawa K, Lokugamage KG, Makino S. Viral and Cellular mRNA Translation in Coronavirus-Infected Cells. Adv Virus Res. 2016;96:165-192. doi: 10.1016/bs.aivir.2016.08.001. Epub 2016 Sep 10. PMID: 27712623; PMCID: PMC5388242.
- Ali Syeda Z, Langden SSS, Munkhzul C, Lee M, Song SJ. Regulatory Mechanism of MicroRNA Expression in Cancer. Int J Mol Sci. 2020 Mar 3;21(5):1723. doi: 10.3390/ijms21051723. PMID: 32138313; PMCID: PMC7084905.
- Han H. RNA Interference to Knock Down Gene Expression. Methods Mol Biol. 2018; 1706:293-302. doi: 10.1007/978-1-4939-7471-9_16. PMID: 29423805; PMCID: PMC6743327.
- Fire A., Xu S., Montgomery M.K., Kostas S.A., Driver S.E., Mello C.C. Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature. 1998; 391:806–811. doi: 10.1038/35888.
- Zamore PD, Tuschl T, Sharp PA, Bartel DP. RNAi: double‐stranded RNA directs the ATP‐dependent cleavage of mRNA at 21 to 23 nucleotide intervals. Cell 2000; 101: 25–33.
- Hammond SM, Bernstein E, Beach D, Hannon GJ. An RNA‐directed nuclease mediates post‐transcriptional gene silencing in Drosophila cells. Nature 2000; 404: 293–296.
- Bernstein E, Caudy AA, Hammond SM, Hannon GJ. Role for a bidentate ribonuclease in the initiation step of RNA interference. Nature 2001; 409: 363–366.
- Ketzinel-Gilad M, Shaul Y, Galun E. RNA interference for antiviral therapy. J Gene Med. 2006 Aug;8(8):933-50. doi: 10.1002/jgm.929. PMID: 16779870; PMCID: PMC7166902.
- Harper SQ, Staber PD, He X, Eliason SL, Martins IH, Mao Q, Yang L, Kotin RM, Paulson HL, Davidson BL. RNA interference improves motor and neuropathological abnormalities in a Huntington’s disease mouse model. Proc Natl Acad Sci U S A. 2005 Apr 19;102(16):5820-5. doi: 10.1073/pnas.0501507102. Epub 2005 Apr 5. PMID: 15811941; PMCID: PMC556303.
- Ma Y, Chan CY, He ML. RNA interference and antiviral therapy. World J Gastroenterol. 2007 Oct 21;13(39):5169-79. doi: 10.3748/wjg.v13.i39.5169. PMID: 17876887; PMCID: PMC4171298.
- Haasnoot J, Berkhout B. RNA interference: its use as antiviral therapy. Handb Exp Pharmacol. 2006;173(173):117-50. doi: 10.1007/3-540-27262-3_7. PMID: 16594614; PMCID: PMC7120273.
- Aagaard L, Rossi JJ. RNAi therapeutics: principles, prospects and challenges. Adv Drug Deliv Rev. 2007 Mar 30;59(2-3):75-86. doi: 10.1016/j.addr.2007.03.005. Epub 2007 Mar 16. PMID: 17449137; PMCID: PMC1978219.
- Gavrilov K, Saltzman WM. Therapeutic siRNA: principles, challenges, and strategies. Yale J Biol Med. 2012 Jun;85(2):187-200. Epub 2012 Jun 25. PMID: 22737048; PMCID: PMC3375670.
- Song E, Zhu P, Lee SK, Chowdhury D, Kussman S, Dykxhoorn DM, Feng Y, Palliser D, Weiner DB, Shankar P, et al. Antibody mediated in vivo delivery of small interfering RNAs via cell-surface receptors. Nat Biotechnol. 2005;23:709–717.
- Rai MF, Pan H, Yan H, Sandell LJ, Pham CTN, Wickline SA. Applications of RNA interference in the treatment of arthritis. Transl Res. 2019 Dec;214:1-16. doi: 10.1016/j.trsl.2019.07.002. Epub 2019 Jul 10. PMID: 31351032; PMCID: PMC6848781.
Figure References:
- Karnati HK, Yalagala RS, Undi R, Pasupuleti SR, Gutti RK. Therapeutic potential of siRNA and DNAzymes in cancer. Tumour Biol. 2014 Oct;35(10):9505-21. doi: 10.1007/s13277-014-2477-9. Epub 2014 Aug 23. PMID: 25149153.
Inspector: Furkan EKER