ABSTRACT
RNA interference is a natural process in which a double-stranded RNA molecule is introduced to a cell. There, it is transformed into short RNA duplexes, causing what is known as gene silencing. siRNA is an example of RNA that can induce this process. It is specific in nature, binding to specific mRNA targets through the Watson-Crick base pairing mechanism. This decreases the emergence of side effects and toxicity, making it a potential candidate for therapeutic use in cancer and viral infection treatment. Silencing of receptors and proteins responsible for cancer cell proliferation is a common strategy in siRNA application in chemotherapeutics. Additionally, siRNA can also be used to sensitize drug-resistant cancer cells. In viral infections, RISC-mediated cleavage of virally encoded cytoplasmic viral mRNA is a mechanism used in siRNA-based antiviral therapy. The vast applications of siRNA prompts further study of more potential uses of siRNA.
Keywords: RNA interference, gene silencing, siRNA, cancer, viral infections.
CHAPTER 1 — INTRODUCTION
Since the discovery of the double helix structure of DNA in 1953, the idea of gene editing has often been regarded as science fiction. However, through the vast advancements in genetic engineering and recombinant technology, what once used to be the far future is now at hands-reach through gene therapy. Gene therapy is defined as the capacity of gene improvement through the correction of altered genes with the intention of therapeutic treatment. Principally, a normal gene is inserted into the genome to replace a target “abnormal” gene.1 In the past 10 years, more than 300 clinical trials involving gene therapy were performed with the involvement of over 3000 individuals. Gene therapy is suited for long-term delivery of transgene to patients suffering from genetic deficiency that is unable to be cured through protein or pharmacokinetic therapy.2 Many methods have been discovered and are currently in place for gene therapy, including CRISPR-Cas9, genetically modified cell-based immunotherapies, and, more recently, RNAi.
RNAi or RNA interference is a natural phenomenon first discovered in Caenorhabditis elegans in 1998. RNAi is a process in which a double-stranded RNA molecule is introduced to a cell where it will be transformed into short RNA duplexes, causing what is known as gene silencing. A type of RNA that is able to undergo this process is small interfering RNA or siRNA. siRNA is a double-stranded RNA (dsRNA) molecule that consists of 19 base pairs with 2 base pairs at the 3’ end that are unpaired.3
There are two stages in RNAi: the initiation phase and execution phase. In the initiation phase, dsRNA in the form of siRNA is split into several RNA fragments 20-25 nt in length with a specific secondary structure. Enzymes known as dicers are able to sense the presence of dsRNA and bind to them through the RNA Binding Domain. Dicer is then able to separate the 2 strands, splitting the siRNA on a nucleotide sequence basis. In the execution phase, siRNA that was split will associate with a multi-protein complex known as the RNA-Induced Silencing Complex (RISC) that consists of argonaute proteins. One of the stands will be designated as the guide strand while the other is removed and degraded. The guide strand has a nucleotide sequence that is complementary to a specific part of the target mRNA, making the process specific. Upon binding to the target mRNA, the mRNA will be split by the RISC and degraded by one of the proteins within the complex.3
The ability of siRNA to specifically target selected nucleotide sequences, this technology holds much anticipation in its use in drug delivery and therapy. Like most gene therapy applications, siRNA therapy is still experimental and has yet to move from the bench to the shelves. Aspects where siRNA can be applied in therapy include cancer therapy, viral infections, cardiovascular diseases, regenerative medicine, and drug delivery enhancement.
CHAPTER 2 — DISCUSSION
Cancer sits on the second place of the most common cause of death worldwide. As of today, it is estimated that there are 2,000,000 cases of cancer and 10,000,000 deaths caused by cancer. Another concomitant threat to humanity is the threat of viruses and viral infections. Infectious disease kills 15 million people each year, contributing 26% of the total annual deaths in the global population. siRNA therapy has the potential to overcome the two. This section discusses the applications of siRNA in cancer therapy and in viral infections.
3.1 Cancer Therapy
The various advancements in cancer research has resulted in extensive profiling of the genome of cancer cells, identifying which part of the genome is responsible for the development of cancer. The common targets of chemotherapeutic drugs are receptors that are abundant within cancer cells but less commonly found in normal cells. These receptors are often responsible for cell signaling or are keys to cell proliferation. siRNA is utilized in cancer therapy to knock-down the genes that are directly or indirectly responsible for the proliferation of cancerous cells.
Although these receptors are easily inhibited by small molecules or other therapeutic agents, these molecules bind with more than 300 non-target proteins. This is responsible for the side effects and toxicity that come with cancer therapy.4 siRNA is specific in nature due to the Watson-Crick base pairing mechanism between siRNA and mRNA. This is because siRNA will only bind to the target mRNA, making it more specific than common chemotherapeutic drugs and thus decreasing the likelihood of side effects and toxicity.5
Multiple studies in vitro and in vivo studies have confirmed the gene-silencing ability of siRNA in cancer treatment. Elongation factor 2 kinase (EF2K) is an enzyme that is overexpressed in patients with breast cancer experiencing mutations in BRCA1, a human tumor suppressor gene. This overexpression leads to significant tumor growth and poor survival rates. The use of siRNAs to target the EF2K enzyme was shown to substantially decrease cell proliferation, migration, and invasion of the cancer cells. In addition to that, the acetylation of ATP-binding cassette transporter E1 (ABCE1) induced by a protein known as Tat interactive protein 60 kDa (Tip60) is increasingly found in tissues and cells of lung cancer patients. siRNA designed to silence Tip60 in lung cancer cells is associated with a decline of ABCE1 acetylation, leading to reduced tumor weight and volume.6
Apart from its use in direct silencing of genes involved in cancer, siRNA has also been used to sensitize drug-resistant cancer cells. Docetaxel is a chemotherapeutic drug that is commonly used in the treatment of prostate cancer. However, it has been found that cancer cells are developing resistance towards the drug. In an effort to increase the efficacy of docetaxel therapy, the potential of gene silencing of Notch-1, an overexpressed transmembrane receptor that is significant in the early development of prostate cancer, was determined. Results showed that Notch-1 silencing using siRNA decreases the proliferation and increases apoptosis of tumor cells. Additionally, Notch-1 silencing increases the sensitivity of PC-3 cells to docetaxel chemotherapy.7
3.2 Viral Infections
The use of siRNA is a flexible approach to the treatment of viral infections. Virus replication is inhibited through RISC-mediated cleavage of virally encoded cytoplasmic mRNAs. siRNA can also be used exogenously to degrade viral RNAs through RNAi pathways. There are two categories of siRNA-based therapeutics against viral infections: targeting of viral proteins necessary for viral growth and replication, and targeting of host factors responsible for the intracellular entry of viruses. The mechanism that underlies the treatment of viral infections using siRNA involves targeting and initiating transcription termination of the most-critical mRNAs that encode essential viral proteins.8
Currently, there are no approved antiviral therapies based on RNAi and siRNA. The first RNAi-based therapy to enter human clinical trials was ALN-RSV01. It consists of a sole siRNA designed to target the mRNA encoding for the nucleocapsid protein of the respiratory syncytial virus.9 Another example of siRNA-based therapeutics is VIR-2218, a GalNAc-conjugated siRNA targeted to reduce the surface antigens of the Hepatitis B virus.10 Although there are many ongoing trials for RNAi-based therapeutics, there needs to be more research on the delivery of such therapeutics as well as the long-term effects of such therapies.
CONCLUSION
The prospect of siRNA in tackling global challenges in terms of mitigating cancer and viral infection cases was discussed in this review. siRNA utilizes RNAi, a natural phenomenon that silences specific gene codons through the RISC and mRNA termination. The specificity of siRNA allows for its use in cancer therapy to diminish the expression of certain receptors and proteins that are responsible for cancer progression. Notably, trials for breast cancer and prostate cancer have demonstrated its use in chemotherapeutics. In viral infections, siRNA is able to initiate the destruction of viral encoded mRNA and prevent viral entry. With the wide range of applications of siRNA-based therapies, this review urges the further study of siRNA and the continuation of current clinical trials.
REFERENCES
1. Gonçalves G A R, Paiva R M A. Gene therapy: advances, challenges and perspectives. Einstein (São Paulo). 2017. Available from:
https://doi.org/10.1590%2FS1679-45082017RB4024
2. Scheller E L, Krebsbach P H. Gene Therapy. Journal of Dental Research. 2009. Available from:
https://doi.org/10.1177%2F0022034509337480
3. Felekkis K, Deltas C. RNA Interference: A powerful laboratory tool and its therapeutic implications. Hippokratia, 10(3): 112-115. 2006.
4. Zhou H, Gao M, Skolnick J. Comprehensive prediction of drug-protein interactions and side effects for the human proteome. Scientific Reports 5. 11090. 2015.
5. Hu B, Zhong L, Weng Y, Peng L, Huang Y, Zaho Y, Liang XJ. Therapeutic siRNA: state of the art. Signal Transduction and Targeted Therapy 5, 101. 2020.
6. Bahreyni A, Luo H. Advances in Targeting Cancer-Associated Genes by Designed siRNA in Prostate Cancer. Cancers (Basel). 2020. Available from:
https://doi.org/10.3390%2Fcancers12123619
7. Ye Q F, Zhang Y C, Peng, X Q, Long Z, Ming, Y Z, He, L Y. Silencing Notch-1 induces apoptosis and increases the chemosensitivity of prostate cancer cells to docetaxel through Bcl-2 and Bax. Oncology Letters. Spandidos Publications. 2012. Available from:
https://doi.org/10.3892/ol.2012.572
8. Alvarez R, Elbashir S, Borland T, Toudjarska I, Hadwiger P, John M, Roehl I, Morskaya S S, Martinello R, Khan J, Ranst M V, Tripp R A, DeVincenzo J P, Pandey R, Maier M, Nechev L, Manoharan M, Kotelianski V, Meyers R. RNA interference-mediated silencing of the respiratory syncytial virus nucleocapsid defines a potent antiviral strategy. Antimicrob Agents Chemother. 2009. Available from: 10.1128/AAC.00014-09
9. Kang H, Ga Y J , Kim S H, Cho Y H, Kim J W, Kim C, Yeh J Y. Small interfering RNA (siRNA)-based therapeutic applications against viruses: principles, potential, and challenges. Journal of Biomedical Science. 2023.
10. Gane E, Lim YS, Kim JB, Jadhav V, Shen L, Bakardjiev AI, et al. Evaluation of RNAi therapeutics VIR-2218 and ALN-HBV for chronic hepatitis B: results from randomized clinical trials. J Hepatol. 2023.
