By Chuyue Zhang
Cancer is one of the most prevalent diseases and the second leading cause of death in the world (Chalbatani et al.,2019). Recently, using RNA interference (RNAi) and RNA activation (RNAa) for cancer treatment is in the research spotlight. RNAi refers to the sequence-specific gene silencing by double-stranded RNA (dsRNA) which is homologous to a specific gene, while RNAa is the opposite process that enhances gene expression (Chalbatani et al.,2019)(Kwok et al.,2019). The corresponding non-coding RNAs associated with these regulation processes are small interfering RNA(siRNA) and small activating RNAs (saRNA).
The mechanisms of siRNA and saRNA are key to successfully making desirable changes in target protein levels to treat cancer.
The RNAi process starts with 21-25 nt long siRNA formation when double-stranded RNAs (dsRNAs) are cleaved by the enzyme dicer. Then siRNA can be recognised by the RNA-induced silencing complex (RISC). The double-stranded RNA will unwind and incorporate the antisense onto RISC. The antisense strand acts as a guide strand to the complementary mRNA sequence and leads to RISC binding to the target mRNA. Argonaute 2(Ago2) will cleave the target mRNA and gene expression is downregulated (Figure 1a) (Chalbatani et al.,2019).
The RNAa mechanism also involves the action of Ago2, but for gene activation, the antisense of saRNA loaded to Ago2 guides the saRNA- Ago2 complex to the corresponding promoter. The binding of the complex can recruit RNA helicase RHA and the RNA polymerase-associated protein CTR9. It leads to chromatin structure opening by histone remodelling enzymes and transcriptional activation by phosphorylated RNA polymerase II (Figure 1b) (Yoon & Rossi, 2018) (Kwok et al.,2019).
(Figure 1a(left): the mechanism of RNAi involving siRNA. dsRNA is unwinded and led to the complementary mRNA by forming a complex with RISC and induce gene silencing by Ago2 cleavage. The process takes place in cytoplasm(Chalbatani et al.,2019).
Figure 1b (right): The process of RNAa involving saRNA. dsRNA is unwinded and antisense strand is loaded to Ago2 in cytoplasm. Then the complex enters the nucleus and bind to the promoter region to induce mRNA transcription of the target gene(Yoon & Rossi, 2018) (Kwok et al.,2019).)
RNAi and RNAa are highly regulated and evolutionary conservative, making them applicable to a wide range of diseases caused by increased or decreased key protein expression, e.g. cancer(Kwok et al.,2019). Also, these small RNAs can easily be synthesised chemically in a high yield, unlike mRNA which is difficult to produce if the length is over 150 nt(Mu et al., 2018).
However, there are still challenges and limitations when using siRNAs and saRNAs as therapeutic tools for cancer, and corresponding strategies to overcome them are being developed.
Small RNAs are often susceptible to nuclease attack and can be very unstable. A common approach to increase nuclease resistance is 2′-O-methyl and 2′-fluoro modifications in the RNA. These modifications can at the same time inhibit the immune stimulatory effect of saRNA. Interestingly, 2′-fluoro modification is only added to the guide strand because it will decrease the potency when both sense and antisense strands are modified. A way to enhance the potency of saRNA is by adding a 5′ inverted abasic modification on the sense strand. This can increase the effectiveness of antisense strand loading in AGO2(Kwok et al.,2019). For siRNA, there have been strategies to replace non-bridging oxygen on the phosphate linkage with a sulfur atom, or modify the 2′-hydroxyl group of the sugar ring with a methyl group (2′- OCH3) and ethyl group (2ʹ-OCH2CH3) to increase its efficacy and potency(Chalbatani et al.,2019).
Another problem to consider when applying siRNAs and saRNAs in vivo is their off-target effect. Thus, the uniqueness of the selected sequence is crucial. Off-target silencing could occur when siRNA binds to three prime untranslated regions (3’-UTR) of the mRNA, which will affect many transcripts instead of the single target. Synthetic siRNAs sometimes also compete with endogenous miRNAs for RISC in the RNAi pathway. This is called RNAi machinery saturation, where there will be a mixed effect of both transcript upregulation and downregulation of target gene(Alagia & Eritja, 2016).saRNA is also proposed to have off-target effects because it shares the same structure as siRNA(Kwok et al.,2019). Reducing off-target effects requires identification of possible off-target transcripts and designing of more effective modified small RNAs.
Other than modifications of the RNAs, different in vivo delivery methods have been extensively researched and developed to increase the treatment’s efficacy. Common delivery methods include using lipid-based delivery vehicles, inorganic nanoparticles, and polymeric nanoparticles like dendrimers and aptamers(Chalbatani et al.,2019). These methods increase the half-life and uptake efficiency of small RNAs by encapsulating them for cellular endocytosis or forming specific structures to better bind to target cells for uptake.
As a revolutionary new class of medicine to treat many previously undruggable cases, siRNA and saRNA have shown promising future applications. For example, there are phase I clinical trials using encapsulated siRNAs in lipid particles and targets PLK1 which is involved in tumor cell proliferation as an approach to treat advanced solid tumours. Previous studies have even shown potential of this treatment in breast, ovarian, and colorectal cancers. Another phase I trial of liver cancer treatment uses two different siRNAs delivered by nanoparticles to target vascular endothelial growth factor (VEGF) and kinesin spindle protein (KSP) (Davidson & McCray, 2011). It is also proposed as a future direction to combine chemotherapeutic agents and siRNA or use several siRNA together targeting different metabolic pathways of cancer(Chalbatani et al.,2019).
For saRNAs, the first saRNA-based drug targeting a transcription factor CEBPA has been shown to decrease liver cancer tumour markers in patients in phase I trial(Reebye et al., 2018). Also, using aptamer-delivered saRNAs can upregulate CEBPA and its downstream targets like cyclin-dependent kinase inhibitors. This has a strong antitumor effect without toxicity in treating pancreatic cancer which is an aggressive type of cancer with no effective treatment currently(Yamamoto et al., 2014) (Yoon et al., 2016). Another use of saRNA is to target CRMP4 that can suppress tumour metastasis in prostate cancer. It has been proven successful in vitro and in orthotopic xenograft nude mice without side effects (Gao et al., 2010).
In conclusion, RNAi involving siRNAs and RNAa involving saRNAs are two complementary processes that regulate protein expression in different directions with some shared proteins(like Ago2). While the pathways still need to be fully studied for RNAi and RNAa, their use in therapeutics to treat diseases like cancer is already showing their versatility and effectiveness. The use of siRNA and saRNA as a less aggressive treatment than classical chemotherapy shows a promising future, while the modification and delivery of the RNAs are still under optimisation to minimise their adverse effects.
References:
Alagia, A., & Eritja, R. (2016). SiRNA and RNAI OPTIMIZATION. Wiley Interdisciplinary Reviews: RNA, 7(3), 316-329. doi:10.1002/wrna.1337
Davidson, B. L., & McCray, P. B. (2011). Current prospects for rna interference-based therapies. Nature Reviews Genetics, 12(5), 329-340. doi:10.1038/nrg2968
Gao, X., Pang, J., Li, L., Liu, W., Di, J., Sun, Q., . . . Li, B. (2010). Expression profiling identifies new function of collapsin response Mediator Protein 4 as A metastasis-suppressor in prostate cancer. Oncogene, 29(32), 4555-4566. doi:10.1038/onc.2010.213
Guo, W., Chen, W., Yu, W., Huang, W., & Deng, W. (2013). Small interfering RNA-based molecular therapy of cancers. Chinese Journal of Cancer, 32(9), 488-493. doi:10.5732/cjc.012.10280
Kwok, A., Raulf, N., & Habib, N. (2019). Developing small activating rna as a therapeutic: Current challenges and promises. Therapeutic Delivery, 10(3), 151-164. doi:10.4155/tde-2018-0061
Mahmoodi Chalbatani, G., Dana, H., Gharagouzloo, E., Grijalvo, S., Eritja, R., Logsdon, C. D.et al., (2019). Small interfering rnas (sirnas) in cancer therapy: A nano-based approach. International Journal of Nanomedicine, Volume 14, 3111-3128. doi:10.2147/ijn.s200253
Mu X, Greenwald E, Ahmad S, Hur S., (2018). An origin of the immunogenicity of in vitro transcribed RNA. Nucleic Acids Res. 46(10), 5239–5249
Reebye, V., Huang, K., Lin, V., Jarvis, S., Cutilas, P., Dorman, S., . . . Habib, N. A. (2018). Gene activation of cebpa using saRNA: Preclinical studies of the first in human saRNA drug candidate for liver cancer. Oncogene, 37(24), 3216-3228. doi:10.1038/s41388-018-0126-2
Reebye, V., Huang, K., Lin, V., Jarvis, S., Cutilas, P., Dorman, S., . . . Habib, N. A. (2018). Gene activation of cebpa using saRNA: Preclinical studies of the first in human saRNA drug candidate for liver cancer. Oncogene, 37(24), 3216-3228. doi:10.1038/s41388-018-0126-2
Yamamoto, K., Tateishi, K., Kudo, Y., Sato, T., Yamamoto, S., Miyabayashi, K., . . . Koike, K. (2014). Loss of histone demethylase kdm6b enhances aggressiveness of pancreatic cancer through downregulation of c/ebpα. Carcinogenesis, 35(11), 2404-2414. doi:10.1093/carcin/bgu136
Yoon, S., & Rossi, J. J. (2018). Therapeutic potential of small activating rnas (sarnas) in human cancers. Current Pharmaceutical Biotechnology, 19(8), 604-610. doi:10.2174/1389201019666180528084059
Yoon, S., Huang, K., Reebye, V., Mintz, P., Tien, Y., Lai, H., . . . Rossi, J. J. (2016). Targeted delivery of c/ebpα -sarna by pancreatic Ductal Adenocarcinoma-specific RNA APTAMERS Inhibits tumor growth in vivo. Molecular Therapy, 24(6), 1106-1116. doi:10.1038/mt.2016.60
Imperial Bioscience Review 